Method for the Generation of Genetically Modified Vertebrate Precursor Lymphocytes and Use Thereof for the Production of Heterologous Binding Proteins

ABSTRACT

The present invention generally relates to the fields of genetic engineering and antibody production. In particular, it relates to the generation of genetically modified vertebrate precursor lymphocytes that have the potential to differentiate into more mature lymphoid lineage cells, and to the use thereof for the production of any heterologous antibody or binding protein.

FIELD OF THE INVENTION

The present invention generally relates to the fields of geneticengineering and antibody production. In particular, it relates to thegeneration of genetically modified vertebrate precursor lymphocytes thathave the potential to differentiate into more mature lymphoid lineagecells, and to the use thereof for the production of any heterologousantibody or binding protein.

BACKGROUND OF THE INVENTION

Monoclonal antibodies have been proven to be effective reagents for bothdiagnosis, prevention and therapy of disease (Glennie and Johnson,Immunol. Today, 21, p. 403-410, 2000). This is due to their uniquecapacity to bind very specifically to particular epitopes on targetmolecules (antigens) via their variable domains and, at the same time,to mediate effector functions via their constant region domains (Frazerand Capra, Fundamental Immunology, W. E. Paul (Ed.), Fourth Edition, p.37-74, 1999) (FIG. 1). This enables monoclonal antibodies tospecifically identify unique antigens in complex mixtures ofmacromolecules and to direct effector functions to these targets.

Antibodies (or immunoglobulins) consist of two identical heavy (H) chainand light (L) chain glycoproteins that are linked via disulphide bonds(FIG. 1). Each H and L chain comprises an N-terminal variable domainthat varies between different antibodies and a C-terminal constantregion, that is identical in different antibodies belonging to the sameimmunoglobulin isotype (FIG. 1). The combination of H and L chainvariable domains generates the antigen binding pocket of the antibodyand determines its specificity (or idiotype), whereas the constantregions determine its isotype (Frazer and Capra, s.a.). The variabilityof immunoglobulins results from the fact that V_(H) and V_(L) domainsare encoded by a multitude of gene segments, that are designated V(variable), D (diversity; only present in the H chain locus), and J(joining) gene segments (Tonegawa, Nature, 302, p. 575-581, 1983) (FIG.1). During the differentiation of B lymphocytes one V, D and J genesegment is randomly selected in each cell for the site-specificrecombination process of V(D)J recombination, that assembles the genesegments, such that new coding regions for V_(H) or V_(L) domains aregenerated (Grawunder et al., Curr. Opin. Immunol., 10, p. 172-180,1998). Due to the multitude of V, D, and J gene segments, andimprecision in gene segment joining, an enormous repertoire of differentV region specificities can be generated by the millions of B lymphocytesproduced by the immune system every day (Melchers et al., Curr. Opin.Immunol., 7, p. 214-227, 1995).

During evolution, immunoglobulin genes have slightly diverged betweendifferent species, such that the constant regions of antibodies differbetween species. As a consequence, immunoglobulins from one species aremost often immunogenic, if introduced into the vascular system ofanother species.

The immunogenicity of xenogeneic monoclonal antibodies therefore limitstheir use in the therapy of human disease, because exposure of patientsto xenogeneic antibodies may result in adverse effects, even acutetoxicity, or might simply lead to the neutralization and clearance ofthe applied antibody, thereby reducing its pharmacological efficacy(Clark, Immunol Today, 21, p. 397-402, 2000) In contrast, administrationof fully human antibodies to patients usually does not lead to any ofthe aforementioned complications.

Human antisera or human polyclonal antibodies have occasionally beenisolated from the blood of individual patients for the treatment orprevention of rare and usually highly lethal diseases, like Ebola virusinfections, or e.g. for the treatment of individuals after exposure tosnake venoms. However, this approach, for many reasons, is impracticalfor the treatment of diseases affecting larger populations. Furthermore,for ethical reasons, it is impossible to immunize humans with a givenantigen for the purpose of monoclonal antibody production, because theB-lineage cells in humans producing the desired antibodies develop andreside in secondary lymphoid organs and cannot easily be obtained.Furthermore, many potential target antigens for therapeutic antibodies,in particular for cancer therapy, are human proteins (Glennie andJohnson, s.a.). Under normal circumstances, humans will not developantibodies against these targets. Therefore, substantial efforts havebeen made in the recent past to develop procedures for the developmentof therapeutic human or humanized antibodies without the need of thehuman immune system.

The simplest approach uses standard genetic engineering techniques forcloning of cDNAs encoding the variable domains of H and L chains (V_(H)and V_(L) domains) from a hybridoma secreting a xenogeneic antibody ofdesired specificity. The cloned variable region cDNAs are then clonedinto appropriate E. coli, yeast, insect or mammalian cell expressionvectors containing human constant region genes. This will allow theproduction of monoclonal antibodies with the xenogeneic V_(H) and V_(L)domains fused to the human C_(H) and C_(L) constant region domains,thereby resulting in a humanized antibody. However, this approach hasthe disadvantage that the fusion of xenogeneic V_(H) and V_(L) domainsto human C_(H) and C_(L) constant regions may result in either decreasedaffinity or altered specificity. In addition, the heterologous V_(H) andV_(L) of the humanized antibody are still immunogenic in humans, becauseframework regions of V domains vary between different species. Thisproblem can in some cases be circumvented by grafting individualcomplementarity determining regions (CDRs), which mediate the directcontact to antigens, onto human framework regions of H and L chainvariable domains (Fiorentini et al., Immunotechnology, 3, p. 45-59,1997). However, for unknown reasons, CDR grafting still often results inthe generation of immunogenetic antibodies and/or the decrease/loss ofaffinity and specificity.

Therefore, two different and more effective approaches have beendeveloped in recent years for the generation of completely humanimmunoglobulins.

The first method is based on the expression (display) of single chainvariable regions (scFv) consisting of one V_(H) and V_(L) domain on thesurface of filamentous bacteriophages of E. coli (Hoogenboom and Chames,Immunol. Today, 21, p. 371-3818, 2000) (cf. also EP 0 585 287 B1, EP 0605 522 B1). Phage display libraries containing a diverse repertoire ofV_(H) and V_(L) chain can be constructed, and individual scFvspecificities can be isolated from such libraries by binding ofrecombinant phages to immobilized antigen (McCafferty et al., Nature,348, p. 552-554, 1990).

Phage display libraries for scFv binding proteins derived from a naturalhuman repertoire have the drawback of being restricted to specificitiescontained in that repertoire, and specificities for many human antigensare therefore most often not represented in these libraries. Althoughcombinatorial or synthetic phage display libraries may be used tocircumvent this problem, a general drawback of this technology is thathigh affinity binding proteins for a given antigen can often not beisolated. Therefore, substantial efforts have been invested inconstructing very large primary libraries by either brute force cloningor site specific recombination procedures.

The best combinatorial or synthetic libraries are estimated to contain10⁹-10¹¹ potentially different binding sites and can yield bindingspecificities in the 1-200 nM range (Hoogenboom and Chames, s.a.).However, higher affinities in the picomolar range (10⁻¹² M), that can bereached during affinity maturation of immunoglobulins in germinal centerB cells in vivo, can only be generated employing additional tediousgenetic engineering procedures, including V gene shuffling, error-pronePCR, the use of E. coli mutator strains etc.

Whatever the outcome of a phage display library selection process is,eventually the genes encoding the scFv binding site need to be reclonedinto suitable expression vectors for human immunoglobulins, and theseneed to be stably transferred into a cellular expression system allowingthe large-scale production of human antibodies, which is anothertime-consuming and expensive procedure.

The second approach for the production of fully human monoclonalantibodies is based on the use of transgenic mice harbouring constructsfor the human immunoglobulin H and L chain gene loci (Jakobovits, Curr.Opin. Biotechnol., 6, p. 561-566, 1995). The human immunoglobulintransgenes are eventually bred onto a genetic background that does nolonger allow assembly of the endogenous murine antigen receptor genes.In these mice, development of B-lineage cells depends on the expressionof immunoglobulin H and L chains from the human transgenic constructs,and B lineage cells of these mice are only capable of producing humanantibodies. There are three variations of this technique. In one, humanimmunoglobulin miniloci are used as transgenes, from which only alimited repertoire of human antibodies can be generated (Fishwild etal., Nat. Biotechnol., 14, p. 845-851, 1996) (also cf. to, EP 0 546 073B1, U.S. Pat. No. 5,874,299, WO. 92/03918). In a second variation, largeregions of the human IgH and IgκL chain gene loci encompassing themajority of the variable region gene segments cloned into yeastartificial chromosomes are used. In a third variation, pieces of humanchromosomes containing the entire human immunoglobulin H and L chaingene loci are integrated into the germline of mice generating so-calledtrans-chromosomic animals. Mice with such complex transgenes develop apractically normal human immunoglobulin repertoire.

Immunization of these transgenic or trans-chromosomic mice results in ahumoral immune response resulting in the generation of fully humanantibodies. This approach of generating human antibodies has oneimportant advantage over the phage display library technology: Highaffinity antibodies for a given antigen can be generated, because thehuman immunoglobulins can undergo affinity maturation in germinal centerB cells, which is a normal process in the course of an immune reaction.

However, there is one important drawback of this technology: First, thegeneration of transgenic or trans-chromosomic mice is very tedious,difficult, time-consuming, and therefore relatively expensive. Forexample, WO 92/03918 discloses B cells of a transgenic mouse expressinghuman monoclonal antibodies. In order to achieve this transgenic mousefertilized eggs are transfected with exogenous elements by introducingthe heterologous IgH and IgL chain transgenes by pronuclear injection.The entire procedure requires the generation of at least four differentmouse strains: two strains with targeted disruptions within theendogenous IgH and IgL chain gene loci, as well as two strains carryingtransgenes or trans-chromosomes for part or all of the human IgH and IgLchain gene loci. In addition, all these four mouse strains have to bebred together, resulting in mice with a genotype carrying homozygousdisruptions of both the endogenous IgH and IgL chain gene loci and atthe same time carrying the human transgenes encoding both theheterologous IgH and IgL chains. Generation of the different knock-outand transgenic mouse strains, their screening and eventual crossbreedingis a lengthy procedure that will require at least two years, even ifeach step is completely optimized. The extensive time-frames requiredfor the development of a xenomouse strain therefore, leaves littleflexibility for designing different mouse strains containing modifiedtransgenic constructs. Therefore, this technology is not suitable forthe modification and improvement of existing antibodies (e.g. of theiraffinity), because this would require the lengthy procedure ofgenerating a novel transgenic mouse strain for this one antibody. Thistechnology is therefore basically restricted to the de novo generationof human antibodies.

Based on the aforementioned facts, there is clearly the need for atechnology allowing the production of fully human antibodies that wouldcombine the advantages of both the phage display system (i.e. speed andflexibility in generating human antibodies, and the ability to modifyand improve the properties of existing antibodies), and of the humanimmunoglobulin transgenic mouse technology (i.e. the ability to obtainhigh affinity antibodies due to affinity maturation occurring in theimmune system, and production of antibodies with physiologic and naturalstructural features).

SUMMARY OF THE INVENTION

The present invention provides means and methods to generate vertebrateprecursor lymphocytes that can be used for the production of any bindingprotein or functional fragment(s) thereof with the ability toselectively bind to an antigen or ligand, including any heterologousantibody, antigen receptor composed of variable domains and constantregions comprising T cell receptors and membrane bound immunoglobulins,any artificial binding protein displaying either wild-type immuneeffector functions or modified or artificial effector functions notderivable from germline encoded heterologous immunoglobulins or antigenreceptors, and any functional fragment(s) thereof. In particular, theinvention provides means and methods to genetically modify vertebrateprecursor lymphocytes and to effect their differentiation into moremature lymphoid lineage cells either in vitro or in vivo, therebygenerating lymphocytes capable of producing said binding protein orfunctional fragment(s) thereof, as well as a method for the productionof said binding protein or functional fragment(s) thereof. Furthermore,the present invention provides genetically modified vertebrate precursorlymphocytes and mature lymphoid lineage cells as well as immortalizedcells derived therefrom suitable for carrying out the methods accordingto the invention.

Thus, the methods of the present invention allow a great deal offlexibility for the generation of lymphoid lineage cells with thepotential to express simply any type of antibody or binding protein,within short periods of time. At the same time, the possibility totransplant these cells into compatible vertebrate hosts, where the cellsparticipate in specific immune functions such as affinity maturation,allows the exploitation of the selectivity of the immune system forgenerating antibodies and immunoglobulin-like or even artificial bindingproteins of high binding affinities for any given antigenic compound orcomposition.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of immunoglobulin structure andgenetics. Antibodies or immunoglobulins (Ig) are glycoproteins that intheir monomeric form are composed of two identical heavy chains (IgH)and two identical light chains (IgL) that are covalently linked bydisulfide bonds. Each IgH chain comprises one N-terminal variable domain(V_(H)) and 3-4 constant region domains (C_(H)1-3, or 4), depending onthe antibody isotype. In addition, antibodies contain a hinge regionbetween the first and the second C_(H) domain, lending flexibility tothe two antigen binding arms of the heterotetrameric glycoprotein. Lightchains consist of only one N-terminal variable (V_(L)) and oneC-terminal constant domain (C_(L)). The arrangement of the two IgH andIgL chains results in a Y shaped monomeric antibody with two highlyvariable binding sites for antigen, which are formed by the associationof the IgH and IgL chain variable domains. Variable domains differ to agreat extent in so-called hypervariable regions, if V-regions arecompared between different antibodies. This variability results from thefact that V regions are not encoded by single genes, but, in the case ofV_(H) regions, by a number of different V, D and J gene segments, fromwhich one each is randomly selected and then recombined to form thevariable coding region of IgH chains (see upper left part of thedrawing). This process is called V(D)J recombination and occurs duringearly B cell development. The IgL chain gene loci contain only a numberof different V and J gene segments, from which coding regions forvariable domains are generated by a random V to J rearrangement (seeupper right part of the drawing). The constant region domains ofantibodies from the same subclass (isotype) determine the effectorfunctions of the antibody, and do not differ between differentantibodies. However, in the course of an immune response, individual Blymphocytes can be induced to change the isotype of the antibody that isexpressed. In this case, a somatic DNA recombination event occursbetween switch regions (indicated by a dot in the graphic representationof the constant region genes in the lower part of the drawing) located5′ of the different constant region genes. For instance, recombinationbetween the μ- and ε-switch regions can result in the deletion of allconstant region genes from Cμ to Cγ4, as indicated in the drawing. As aresult from this, a VDJ variable coding region is brought into proximityto a Cε constant region, leading to the expression of an antibody of IgEisotype.

FIG. 2 illustrates B cell development in the mouse. The differentiationof B lymphocytes in the mouse can be subdivided into distinct stagesthat can phenotypically and genotypically be subdivided. The earliestcommitted precursor B (preB) cells that can be found in the bone marrowof wildtype mice are derived from a pluripotent hematopoietic stem cell(HSC, left), that does not express lineage specific markers (which isdesignated as Lin⁻), but is characterized by the surface expression ofstem cell antigen 1 (Sca-1). From this, preB cells are generated thatusually carry DJ_(H) rearrangements on both IgH chain alleles and thatare characterized by surface expression of B220 (a pan-B cell marker)and c-kit. At this stage all other Ig gene loci are usually still ingermline (GL) configuration. DJ_(H) rearranged preB cells are designatedpreB-1 cells and can be expanded in tissue culture under specificconditions. The next rearrangement event in these cells involves one ofthe V_(H) gene segments that rearranges to a pre-existing DJ_(H)element. If this leads to a productive rearrangement on any allele suchthat a μH chain can be expressed, these cells receive a proliferativesignal, expand and differentiate into preB-II cells. These cells looseexpression of the c-kit surface marker, but gain expression of CD25. Afurther productive rearrangement on any of the IgL chain alleles resultsin the differentiation to immature B cells expressing membrane boundIgM. These immature, naïve B cells can leave the bone marrow, migrateinto peripheral lymphoid organs, where they might eventually encounterantigen. In the course of an immune response, these B cells can thendifferentiate into antibody secreting plasma cells, during which anotherDNA recombination process (class switch recombination) may occur in theIgH constant region locus resulting in the change of antibody isotypethat is secreted by the plasma cells.

FIG. 3 is a schematic overview of the method according to the inventionfor the generation of humanized precursor B lymphocytes (HupreB's).Murine DJ_(H) rearranged preB-I cells can be isolated from fetal liverof mice 14-19 days post coitum, or from the bone marrow of adult mice(Step 1). The long-term culture of these cells requires special tissueculture conditions, including stromal cell feeder layers and/or thepresence of certain growth factors, such as e.g. IL-7 (interleukin 7).Under these conditions, preB-I cells do not differentiate further (Step2). In order to use these cells for the production of heterologousantibodies, it must be prevented that further productive endogenous Iggene rearrangements can be performed in these cells. This can e.g. beaccomplished by introducing cis-acting mutation(s) on both alleles ofthe IgH and IgL chain gene loci (Step 3b), or, alternatively, byintroducing trans-acting mutation(s) that ablate further V(D)Jrecombination of Ig genes, e.g. by deleting one of the two recombinationactivating genes, RAG-1 or RAG-2 (Step 3a). The latter cells can then bestably transduced with recombinant retroviruses capable of mediatingexpression of xenogeneic, in this case human, IgH and IgL chains (Step4a). These retroviral vectors can also be used for the stabletransduction of the former cells carrying cis-acting mutations inendogeneous Ig gene loci. In addition, preB cells carrying cis-actingmutations are still competent for Ig gene rearrangements. It istherefore possible to stably transfect these cells with heterologousgermline Ig gene loci, which may undergo V(D)J rearrangements, and fromwhich novel xenogeneic antibodies can be produced (Step 4b). If theheterologous or xenogeneic IgH and IgL chain genes are from humanorigin, the preB-I cells will only have the potential for the expressionof fully human antibodies, and are therefore referred to as HupreBcells.

FIG. 4 is a schematic representation illustrating the targeting ofendogenous gene loci using positive-negative gene targeting vectors. Forthe targeting of endogenous gene loci, a genomic region of approximately1 kb upstream (short arm) of that locus and a region of approximately 3kb (long arm) downstream of that gene is cloned into an emptypositive-negative targeting vector. As an example, the targetingstrategy for a DJ_(H)3 rearranged IgH chain locus is depicted. Thisconstruct is transfected into preB-I cells and integration of thetargeting vector by homologous recombination is selected and eventuallyscreened for. The positive selection marker allows selection for thestable integration of the construct into the genome of the transfectedcell. The expression of the negative selection marker would be toxic forthe cell, and cells will only survive, if the negative selection markeris lost upon stable integration, which occurs by two homologousrecombination events at the correct locus. Two possible sites forhomologous recombination between the endogenous gene locus and thetargeting construct are indicated by hatched lines. If integration ofthe construct by such homologous recombination events occurs, thepositive drug selection marker replaces the endogenous region located inbetween the short and long arms of homology. If the positive selectionmarker is flanked by two loxP recombination signals, as indicated here,transient expression of cre recombinase can mediate the deletion of theselection marker, such that the same targeting construct can be used fortargeting of the second allele of the same gene locus.

FIG. 5 a illustrates the cloning strategy for the construction of an IgHchain gene locus targeting vector. The detailed cloning strategy for theconstruction of one possible targeting construct for DJ_(H) rearrangedmurine IgH chain gene loci is depicted here at the example of aD_(FL16.1)-J_(H)4 rearranged gene locus. Other DJ_(H) rearranged IgHchain gene loci can be targeted with the same targeting vector. Uniquerestriction sites are indicated in the endogenous gene locus (topconstruct) and the regions of 1523 and 3215 by that are PCR cloned fromgenomic DNA into the empty positive-negative targeting vectorpBSL-flneo-DT4 are indicated (see FIG. 6 b, pBSL-flneo-DT4 was chosen asone example of various different empty targeting vectors). The PCRprimers for the short and the long arms are designed to contain Not Iand Asc I restriction sites for cloning of the fragments into the uniqueand compatible restriction sites in pBSL-flneo-DT4. The organisation ofthe final targeting construct is depicted on the bottom, and positionsof selected restriction sites are indicated.

FIG. 5 b illustrates the cloning strategy for the construction of anIgκL chain gene locus targeting vector. The detailed cloning strategyfor the construction of one possible targeting construct for murine IgκLchain gene loci designed for the targeted deletion of all germline J_(κ)gene segments is depicted here. Unique restriction sites are indicatedin the endogenous gene locus (top construct) and the regions of 739 and3379 by that are PCR cloned from genomic DNA into the emptypositive-negative targeting vector pBSL-flneo-DT4 are indicated (seeFIG. 6 b, pBSL-flneo-DT4 was chosen as one example of various differentempty targeting vectors). As in FIG. 5 a, PCR primers for the short andthe long arms are designed to contain Not I and Asc I restriction sitesfor cloning of the short and long arms into compatible restriction sitesof pBSL-flneo-DT4. The organisation of the final targeting construct isdepicted on the bottom, and positions of selected restriction sites areindicated.

FIG. 5 c illustrates the cloning strategy for the construction of a RAGgene locus targeting vector. The detailed cloning strategy for theconstruction of one possible targeting construct for the murine RAG genelocus designed for the targeted deletion of the RAG-1 gene is depictedhere. The RAG gene locus in the mouse is located on chromosome 2 andcontains the two very closely linked genes, RAG-1 and RAG-2, that areeach essential for Ig gene rearrangements. The open reading frames(ORFs) of both genes are encoded by one single exon, but a shortuntranslated exon downstream of each promoter element (indicated bytriangles) is spliced to one large exon containing the entire RAG ORFs.Unique restriction sites are indicated in the endogenous gene locus (topconstruct). Genomic regions of 1078 and of 2950 by up and downstream ofthe RAG-1 ORF, respectively, are PCR cloned from genomic DNA into theempty positive-negative targeting vector pBSL-flneo-DT4, as indicated(see FIG. 6 b for pBSL-flneo-DT4, which was chosen as one example ofvarious different empty targeting vectors). As in FIG. 5 a, PCR primersfor the short and the long arms are designed to contain Not I and Asc Irestriction sites for cloning of the short and long arms into compatiblerestriction sites of pBSL-flneo-DT4. The organisation of the finaltargeting construct is depicted on the bottom, and positions of selectedrestriction sites are indicated.

FIG. 6 a shows the detailed cloning strategy for DT-α and DT-α(tox176)based positive-negative vectors for gene targeting (preparatory steps).A prerequisite for the generation of empty targeting vectors is toextend the multiple cloning site (MCS) of a regular cloning plasmid,like pBluescript, such that additional useful restriction sites areadded to the polylinker, which can then be used for the insertion ofexpression cassettes for positive and negative selection markers, aswell as for genomic regions required for the targeting of an endogenousgene locus by homologous recombination. Depicted here is the insertionof an additional oligomer with additional restriction sites into the MCSof pBluescript, thereby generating plasmid pBSL with an extendedpolylinker. This construct is used to insert a β-actin promoter drivenexpression cassette for either wild-type diphtheria toxin α or anattenuated version thereof, denoted DTα(tox176) (cf. example 5b, step2). These two constructs with extended multiple cloning site aredesignated pBSL-DT4 (bottom, left) and pBSL-DT4tox176 (bottom, right),respectively. The diphtheria toxin expression cassettes are cloned intopBSL using XbaI and BglII restriction sites, as indicated. A diagnosticNheI restriction site that is unique for the attenuated DTtox176expression cassette is indicated as well.

FIG. 6 b illustrates the detailed cloning strategy for DT-α andDT-α(tox176) based positive-negative vectors for gene targeting (finalcloning steps). Cloning of empty positive-negative targeting vectorscontaining either neomycin, hygromycinB, or puromycin as positiveselection markers and a wild-type diphtheria toxin α expression cassetteare described. The same cloning strategies can be used for vectorscontaining an expression cassette for DT-α(tox176) instead. Restrictionenzymes used for cloning of fragments and linearization of constructsare indicated. All positive selection markers are designed to be flankedby loxP sites (floxed) which can be recognized by cre recombinase. Whilea floxed neomycin expression cassette can be recloned from the existingvector pGL2-neo(m)+(DSM 14705), floxed hygromycinB and floxed puromycinexpression cassettes needed to be inserted in two steps. For this, asynthetic DNA oligomer containing two loxP sites separated by a MluIsite was inserted into pBSL-DT4 in order to generate pBSL-DT4-2loxP, asindicated. In a final step, expression cassettes for hygromycinB andpuromycin resistance were PCR cloned into the unique MluI site ofpBSL-DT4-2loxP.

FIG. 7 a shows the schematic organization of retroviral expressionvectors for human IgH chains. Recombinant retroviral vectors allowingthe expression of human IgH chain proteins exhibit a modular designcontaining Ig locus promoter and enhancer elements, as well as codingregions for the variable and constant region domains of IgH chains. Thetranscriptional orientation of the IgH expression cassette is oppositeto the retroviral 5′LTR promoter element in order to confer a B cellspecific expression pattern based on the Ig specific promoter andenhancer elements. The expression cassette contains a murine V_(H)promoter a leader (L) sequence with VDJ exons encoding IgH variabledomains, the murine intron heavy chain enhancer (E_(μ)H), exons encodingthe constant regions of heterologous IgH chains, exons for the membranespanning regions of immunoglobulins, and elements from the murine IgH3′α enhancer. Different L-VDJ regions, and even libraries of L-VDJregions may be cloned into the unique BamHI and HindIII restrictionsites of the IgH expression vector using restriction enzymes generatingcompatible overhangs. Due to the modular design, constant region exonsor enhancer elements may be replaced using simple molecular biologytechniques (as indicated).

FIG. 7 b shows the schematic organization of retroviral expressionvectors for human IgκL chains. Like the vectors depicted in FIG. 7 a,recombinant retroviral vectors allowing the expression of human IgκLchain proteins exhibit a similar modular design containing IgL chainspecific locus promoter and enhancer elements, as well as coding regionsfor the variable and constant region domains of IgκL chains. Thetranscriptional orientation of the IgκL expression cassette is oppositeto the retroviral 5′LTR promoter element, in order to confer a B cellspecific expression pattern based on the Ig specific promoter andenhancer elements. The expression cassette contains a murine Vκpromoter,a leader (L) sequence with VJ exons encoding IgκL variable domains, themurine κ intron enhancer (κiE), exons encoding the constant region ofheterologous IgL chains, and elements from the murine Ig κ3′ enhancer(κ3′E), as indicated. In addition, retroviral IgκL chain expressionvectors contain a complete puromycin expression cassette, allowing theselection of stably transduced preB cells. Different L-VJ regions, andeven libraries of L-VJ regions may be cloned into the unique BamHI andHindIII restriction sites of the IgH expression vector using restrictionenzymes generating compatible overhangs. Due to the modular design,modified constant region exons or enhancer elements may be replacedusing simple molecular biology techniques (as indicated).

FIG. 8 illustrates the procedure for the retroviral transduction ofmurine stromal cell/IL7 dependent preB cells with retroviral vectorsencoding heterologous IgH and IgL chains. Long-term proliferatingstromal cell/IL7 dependent preB cell lines with mutations interferingwith endogenous Ig gene rearrangements (cf. FIG. 3) can be sequentiallytransduced with retroviral expression vectors encoding IgL chains andIgH chains, as indicated. After successive transduction with firstheterologous IgL and then IgH chain expressing retroviral constructs,the cells have the potential to express heterologous antibodies upondifferentiation of the cells either in vitro, or in vivo (see FIG. 10).

FIG. 9 is a schematic overview of sources, from which variable domaincoding regions can be isolated. Variable domain coding regions can beisolated from many different sources, as indicated in this figure. Themodular design of the retroviral vectors allows the cloning ofretroviral vectors encoding e.g. only a single specificity, for instancefrom an existing useful monoclonal antibody. Upon transduction of thesevectors and transplantation of transduced cells into mice in vivo (seeFIG. 10), single specificities may then be subjected to affinitymaturation. Alternatively, complete VDJ and VJ libraries may be isolatedfrom peripheral blood lymphocytes (PBLs) from either healthy, sick orimmunized patients and then cloned into IgH and IgL chain retroviralexpression vectors. In fact, it is even possible to generate completelysynthetic V_(H) and V_(L) region repertoires, that may be cloned intoretroviral Ig expression vectors, which can subsequently be stablytransduced and functionally selected in vivo (see FIG. 10).

FIG. 10 illustrates transplantation of HupreBs into immunodeficient micefor the production of fully human monoclonal antibodies. HupreB cells,like normal murine preB cells, can be transplanted into murine hosts,where they are able to participate in immune responses of the host. IfHupreB cells are transplanted into immunodeficient hosts lackingendogenous B cells, the only antibodies that can then be produced areheterologous (human) antibodies from the transplanted HupreB cells.There are many different host strains that may be used for thetransplantation of HupreB cells. Strains that are devoid of both B and Tlymphocytes are e.g. the RAG-1- and RAG-2-deficient and scid mousestrains. In order to be able to elicit a T cell dependent immuneresponse involving B lymphocytes derived from transplanted HupreB cells,T helper lymphocyte populations can be isolated and co-transplanted intothese mice (left side). Alternatively, mouse strains lacking only Blymphocytes, like e.g. J_(H) deficient mice, can be reconstituted bytransplantation with HupreB cells alone (right side). Once theperipheral B and T lymphocyte compartments are reconstituted aftertransplantation, these mice can be immunized using convenientimmunization protocols against any desired antigen. Plasma cells derivedfrom these mice can then be immortalized, e.g. by fusion with myelomacells, in order to produce (hybridoma) cells capable of permanentlysecreting heterologous (human) monoclonal antibodies specific for theinjected immunogenic compound or composition.

FIG. 11 a illustrates the construction of a retroviral expression vectorfor constitutive expression of the anti-apoptotic gene bcl-2(preparatory steps). Removal of IL-7 from continuously proliferatingpreB cell cultures in vitro results in the differentiation of preBcells, but at the same time also leads to the induction of apoptosis.This apoptosis can be prevented by constitutive expression of ananti-apoptotic gene, like e.g. bcl-2. For the generation of a selectableretroviral expression vector for bcl-2, the murine bcl-2 cDNA issequentially cloned with an hygromycin B open reading frame into a dualgene expression vector pIRES. This vector contains an internal ribosomalentry sequences flanked by two multiple cloning sites, into which twodifferent genes can be cloned. This allows the simultaneous expressionof two genes in mammalian cells from one single promoter. The strategyfor the insertion of the bcl-2 ORF and the hygromycin B selection markergene into pIRES for the generation of a pBcl2-IRES-hygroB vector isindicated.

FIG. 11 b illustrates the construction of a retroviral expression vectorfor constitutive expression of the anti-apoptotic gene bcl-2 (finalcloning step). After assembly of the CMV promoter drivenbcl-2-IRES-hygromycinB cassette, the entire dual expression cassettefrom the pBcl2-IRES-hygroB vector is recloned as a SalI-ClaI fragmentinto the retroviral transfer vector pLIB, as depicted. This generatesthe recombinant retroviral construct pLIB-Bcl2-IRES-hygroB that can beused to stably transduce preB cells and to confer simultaneousexpression of bcl-2 and resistance to hygromycinB.

FIG. 12 a exemplifies the construction of retroviral expression vectorsfor the expression of human IgH chains (preparatory step I). The variousIgH promoter and enhancer elements, as well as the coding regions forthe different variable and constant region domains, are sequentiallycloned into a retroviral transfer vector, like e.g. plasmid pLIB. Thisplasmid contains all elements required for retroviral packaging andprovirus integration, i.e. a 5′ LTR, a packaging signal, and a 3′ LTR,cloned into a bacterial, ampicillin selectable plasmid backbone with aColE1 origin of replication (as indicated). Depicted here are the firstcloning steps for inserting a V_(H) promoter, the murine heavy chainintron enhancer (EμH), including flanking matrix attachment regions(MAR) and the IgH chain 3′α enhancer (3′αE). Restriction enzymesincorporated at the termini of PCR amplified fragments, as well asunique compatible restriction enzyme sites in the plasmid backbone,which can be used in the various cloning steps, are highlighted.

FIG. 12 b exemplifies the construction of retroviral expression vectorsfor the expression of human IgH chains (preparatory step II). The IgH3′α enhancer contains four functional regions, which upon activation ofthe enhancer during terminal B cell differentiation become sensitive toDNasel digestion and are therefore referred to as hypersensitive regionsHS 1, 2, 3, and 4. Each of these HS regions contributes to the enhanceractivity of the 3′αE, but most of the enhancer activity is conferred bythe HS1,2 regions. Although the HS1,2 region of the 3′αE alone is ableto drive reporter gene expression to almost the same levels as theentire 3′αE region, it may be desirable for some retroviral IgHexpression constructs to include the other functional regions of the3′αE. Therefore, the cloning of the HS3 and HS4 regions of the 3′αE isdepicted here. Restriction enzymes incorporated at the termini ofgenomic PCR fragments, as well as compatible restriction enzyme sites inthe plasmid backbone, which can be used in the various cloning steps,are highlighted.

FIG. 12 c shows the construction of retroviral expression vectors forthe expression of human IgH chains (preparatory step III). Following thecloning of IgH promoter and enhancer elements into a retroviral transfervector, coding regions for IgH chains need to be inserted. In order tobe able to express membrane bound, as well as secreted forms ofimmunoglobulins, exons for the membrane spanning regions of IgH chainsneed to be included in the retroviral expression vectors. The membranespanning regions differ significantly between different isotypes inlength, primary sequence and exon/intron structure. Therefore, differentmembrane spanning regions for IgM, IgG, IgA and IgE heavy chains need tobe cloned. As an example, the PCR cloning and insertion of the fourrespective different membrane spanning regions into plasmid pAB-3 isdepicted. The same cloning procedures can be performed with vectorspAB-4 and pAB5, containing extended versions of the IgH 3′αE.Restriction enzymes incorporated at the termini of genomic PCRfragments, as well as compatible restriction enzyme sites in the plasmidbackbone, which can be used in the various cloning steps, arehighlighted.

FIG. 12 d illustrates the construction of retroviral expression vectorsfor the expression of human IgH chains (preparatory step IV). Human Blymphocytes are able to produce four different IgG isotypes, IgG1, IgG2,IgG3, and IgG4, that differ slightly in sequence and that exhibitdifferent physiologic effector functions. The cloning of the constantregion genes into an intermediate vector pAB-3.2, already containing IgGmembrane spanning exons, is depicted here. The endogenous exon/intronorganisation including the exons encoding the hinge (H) regions for thevarious IgG isotypes is indicated. Restriction enzymes incorporated atthe termini of genomic PCR fragments, as well as unique compatiblerestriction enzyme sites in the plasmid backbone, which can be used inthe various cloning steps, are indicated. Upon cloning of a VDJrearranged variable domain exon, these constructs are capable ofexpressing complete IgG chains (see FIG. 15).

FIG. 12 e shows the construction of retroviral expression vectors forthe expression of human IgH chains (preparatory step V). The cloning ofconstant region genes for the remaining human Ig isotypes IgM, IgA1,IgA2, and IgE is depicted here. Similar to the cloning procedure for IgGconstant region genes, depicted in FIG. 12 d, the genomic regions forthese isotypes are inserted as NotI digested PCR fragments into uniqueNotI restriction sites upstream of the respective membrane spanningexons for these isotypes. The endogenous exon/intron organisationincluding the exons encoding the hinge (H) regions for the various Igisotypes is indicated. Restriction enzymes incorporated at the terminiof genomic PCR fragments, as well as unique compatible restrictionenzyme sites in the plasmid backbone, which can be used in the variouscloning steps, are indicated. Upon cloning of a VDJ rearranged variabledomain exon, these constructs are capable of expressing complete IgHchains of the respective isotype (see FIG. 15).

FIG. 13 a shows the construction of retroviral expression vectors forthe expression of human IgκL chains (preparatory step I). Similar to theconstruction of retroviral expression vectors for IgH chains, expressionvectors for IgκL chains require the κL chain locus specific promoter andenhancer elements in addition to the coding regions for the κL chainproteins, which need to be sequentially cloned into a retroviraltransfer vector. Starting from the basic retroviral vector pLIB, first,a Vκ promoter is cloned into pLIB, as indicated. This is followed by theinsertion of a PCR product containing the human κ intron enhancer (κiE,with its upstream matrix associated region, MAR) and the human κL chainconstant region gene (left side of the drawing), or, alternatively, bythe insertion of a fusion PCR fragment of the mouse κiE with the humanκL chain constant region gene (right side of the drawing), which isgenerated by a single overlap extension (SOE) PCR. Into both constructs,a PCR product containing the murine κ3′ enhancer (κ3′E) is inserted.Restriction enzymes incorporated at the termini of the various genomicPCR fragments, as well as unique compatible restriction enzyme sites inthe plasmid backbone, which can be used in the various cloning steps,are indicated.

FIG. 13 b illustrates the construction of retroviral expression vectorsfor the expression of human IgκL chains (preparatory step II).Retroviral expression constructs for human IgH and IgκL chains will besequentially transduced into preB cells, and it is therefore desirableto be able to select for stably transduced cells after the first roundof transduction with IgκL chain expressing vectors. For this reason aSV40 promoter driven puromycin expression cassette is inserted into thetwo possible IgκL chain retroviral transfer vector intermediates pAB-7.1and pAB-8.1, as indicated. Upon cloning of a VκJκ rearranged variabledomain exon into these vectors, these retroviral constructs are capableof expressing human IgκL chains (see FIG. 15).

FIG. 14 a shows the construction of retroviral expression vectors forthe expression of human IgλL chains (preparatory step I). Retroviralexpression constructs for human IgλL chains are cloned by firstinserting a fusion PCR product of a puromycin cassette and a Vλ promoterinto pLIB (the PCR fusion is performed by single overlap extension (SOE)PCR as described in the example section). In the next step, a SOE-PCRfusion construct of two murine λL chain enhancer elements, the λ2-4 andthe λ3-1 enhancer, are inserted into the construct, as indicated.Finally, the coding region of the human Cλ1 region is inserted by PCRcloning into this retroviral cloning vector using the indicatedrestriction enzymes. Upon cloning of a VλJλ rearranged variable domainexon into these vectors, these retroviral constructs are capable ofexpressing human IgλL chains (see FIG. 15).

FIG. 14 b illustrates the construction of retroviral expression vectorsfor the expression of human IgλL chains (preparatory step II). Otherhuman IgλL chain constant region genes may be used to replace the Cλ1coding region by simple cloning using the unique MluI-NotI restrictionenzymes, as indicated. Upon cloning of VλJλ rearranged variable domainexons into these vectors, these retroviral constructs are capable ofexpressing the different human IgκL chain isotypes, as indicated (seeFIG. 15).

FIG. 15 a shows the insertion of V-region genes into retroviral humanIgH, IgκL and IgλL chain expression vectors. All retroviral expressionconstructs for human IgH and L chains that have been described so fardid not contain coding regions for the variable domains. These can beisolated by PCR cloning from various sources (see e.g. FIG. 9) and maybe inserted into the retroviral vectors as V region exons encoding asingle specificity, or as libraries encoding different specificities(which need to be PCR amplified with degenerate primer pairs). V regionsare PCR amplified together with their characteristic leader (L)sequences, as indicated. Restriction enzymes that can be used for thecloning of variable region exons are indicated. This drawing depictssome selected final retroviral IgH and IgL chain expression vectors. Dueto the modular design of these vectors, different coding regions andpromoter and enhancer elements may be used.

DEFINITION OF TERMS USED THROUGHOUT THE SPECIFICATION

Affinity: The strength of the binding of an antigen receptor (or of anyreceptor) for its cognate antigen (or its ligand or binding partner),determined by the ratio of association to dissociation rate of thereceptor-ligand interaction.

Affinity maturation: A highly regulated immunological process of antigendriven improvement of the binding specificities of antibodies producedby antigen stimulated B lymphocytes in germinal centers. The process iscaused by somatic hypermutation within the coding regions for thevariable domains of antibodies coupled with the selective expansion andsurvival of B lymphocytes generating higher affinity antibodies.

Antibody: A glycosylated polypeptide produced by B lymphocytes andplasma cells comprising in its monomeric form two identical heavy (H)chains and two identical light chains, each being composed of onevariable domain and one (L chains) or several (H chains) constant regiondomains. The two H and two L chains assemble into a symmetric Y shapeddisulphide linked antibody molecule that has two binding domains formedby the combination of the variable regions of H and L chains.

Antigen: Any biomolecule or chemical entity that can be bound by thevariable domains of immunoglobulins (or antibodies).

Antigen receptor: Antigen receptors are composed of variable domains andconstant regions, the former having the ability to bind to antigens, andthe latter to mediate effector functions or to transduce signals intocells. Antigen receptors comprise T cell receptors and membrane boundimmunoglobulins.

Artificial binding protein: A binding protein containing polypeptidesequences (e.g. synthetic spacer sequences) not derivable from naturalgenes or fragments thereof.

Binding protein: The most general term for any type of polypeptide withthe ability to selectively bind to an antigen or ligand, with or withoutadditional effector functions. Binding proteins include artificialbinding proteins, fragments of antibodies, like e.g. a single chainvariable fragment (scFv), and also fusion products combining variableand constant region domains of immunoglobulins and T cell receptors, orvice versa. Furthermore, a binding protein can be the fusion product ofvariable domains with functional parts of other receptor molecules.Conversely, a binding protein can be the fusion of a binding moiety froma non-Ig or TCR related receptor with constant region domains of antigenreceptors.

Cis-acting: Having an influence only on genetic elements and gene lociin vicinity or on the same allele.

Coding region: A genetic element that has an open reading frame that canbe expressed into a polypeptide.

Complementary determining regions (CDRs): The regions in the threedimensional structure of a variable antigen receptor domain thatdirectly establish contact to antigens. The CDRs are usually the mostdiverse parts of antigen receptors.

Domain: A structural moiety of a biomolecule that is characterized by aparticular three dimensional structure (e.g. variable or constant regiondomains of immunoglobulins, that are structurally related, such asIg-like domains that can be found in many molecules of the immunesystem, which belong to the so-called Ig-superfamily).

Effector functions: Functions of constant regions of immunoglobulins inthe context of the immune system, e.g. the ability to activatecomplement, or to activate certain immune cells by binding to specificreceptors for constant region (F_(c) receptors).

Endogenous: Being self to a certain cell or organism.

Enhancer: A genetic element that can stimulate the activity of promoterelements over large distances, independent of orientation or position.

Epitope: A three dimensional structural entity of an antigen that isrecognized by a variable binding region of an antibody.

Functional fragment (of an antibody, antigen receptor or bindingprotein). A functional fragment derivable from a given polypeptideselected from the group consisting of antibodies, antigen receptors andbinding proteins, that shares at least one of the desired propertiesinherent to said polypeptide with respect to the specific field ofapplication. For example, a functional fragment may be a polypeptide oroligopeptide maintaining the capability of said polypeptide to bind tocertain antigens and/or ligands. The same applies to fragments mediatingspecific effector functions characteristic for said polypeptide. Afurther example relates to fragments being able to activate or inhibitspecific immunogenic and/or physiologic effector functions ofantibodies, antigen receptors and regular receptor-ligand systems.

Genetic elements: Genes, gene loci, or fragments thereof, likepromoters, enhancers, and coding regions, alone or in any functional ornon-functional combination.

Germinal center: A distinct histological structure in peripherallymphoid organs (e.g. lymph nodes or spleen) where cognate interactionsbetween antigen presenting cells and different lymphocyte populationsoccur, resulting in the proliferative expansion of antigen reactivelymphocytes, as well as affinity maturation and class switchrecombination of antibodies produced by antigen reactive B lymphocytes.

Germline configuration: The native configuration of genes and gene loci,as they are inherited from the parents, and as they will be passed on tofurther generations through the germline. DNA recombination eventsoccurring in somatic cells, like e.g. V(D)J recombination inlymphocytes, lead to the reshuffling or loss of genetic information oncertain gene loci and therefore to a change of the genes from thegermline configuration.

Heterologous: Being different from endogenous.

Humanized antibody: An artificial antibody generated by fusing thevariable domains of non-human antibodies to human constant regiondomains.

Hybridoma: An immortal cell line that has been generated by the fusionof an immortal myeloma cell line (unable to secrete antibody) and amortal plasma cell (that may secrete large amounts of antibodies).

Idiotype: The binding characteristic (specificity) of an antigenreceptor.

Isotype: The structural characteristic conferred to by the constantregion of an antigen receptor determining their (effector) functions.

Immunoglobulin (Ig): Synonymous for antibody.

Immunoglobulin minilocus: An artificial genetic construct comprising few(i.e. single digit) V, D and/or J gene segments, capable of undergoingV(D)J recombination, thereby generating a limited, oligoclonalrepertoire of variable coding regions.

Library: A collection of different genetic elements.

Monoclonal antibody: Antibody with one defined specificity determined bythe unique structure of the variable antigen binding region of theantibody.

Myeloma cell: An immortal cell derived from a cell at plasma celldifferentiation stage without the ability to express endogenousimmunoglobulin proteins.

Polyclonal antibodies: A mixture of antibodies having differentstructures of the variable antigen binding regions, and therefore, mostlikely, different binding specificities.

PreB lymphocyte: A precursor B lymphocyte is characterized by theexpression of lymphoid specific factors involved in V(D)J recombination(e.g. RAG-1, RAG-2), usually has initiated V(D)J recombination on atleast one immunoglobulin heavy chain allele, but still carries the lightchain gene loci in germline configuration, and therefore does notexpress complete antibodies.

Primary lymphoid organs: Organs, in which lymphocytes develop fromhematopoietic stem cells, in mice and humans e.g. the bone marrow, thethymus, and during fetal life, the liver.

Repertoire: A collection of different (binding) specificities.

Somatic hypermutation: A process in somatic cells resulting in theintroduction of point mutations into specific regions of the genome at ahigh frequency (>10⁻⁴ mutations per basepair per cell division).

Stromal cells: Proliferating adherent cells derived from the bone marrowwith the capacity to support proliferation of preB cells in the presenceof additional preB cell growth factor(s).

Trans-acting: Having an influence on genetic elements and gene locilocated on different chromosomes.

Transfecting: The process of introducing nucleic acid sequences intoeukaryotic cells, usually associated with using chemical and physicalmethods.

Transforming: The process of immortalizing a cell for the establishmentof a continuously proliferating cell line.

Transgene: An artificial genetic element introduced into the germline ofanimals. The transgene is therefore inherited from one generation to theother.

Transducing: The process of delivering DNA into mammalian cells via theproduction of recombinant viruses. For this, a packaging cell line,expressing structural proteins for viral particles is transfected with arecombinant viral DNA construct comprising the regulatory elements forpackaging of the viral DNA construct into the viral structural proteins.By this, recombinant viruses are produced that can be used to infectmammalian target cells leading to the introduction of the geneticinformation cloned into the recombinant viral genome.

V(D)J recombination: A DNA recombination process during which one ofmany V(variable), sometimes D (diversity), and J (joining) gene segmentsare assembled to a coding region for the variable domains of antigenreceptors.

Vector: An artificially generated nucleic acid construct which can beused to shuttle nucleic acid elements between different organisms andspecies, and which can further be used to propagate, amplify andmaintain genomic information.

Xenogeneic: Being derived from a different species (often usedsynonymous to heterologous).

All references cited throughout the specification are incorporatedherewith.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the present invention provides a method forthe generation of vertebrate lymphocytes that can be used for theproduction of any binding protein or functional fragment(s) thereof withthe ability to selectively bind to an antigen or ligand, including anyheterologous antibody, any antigen receptor composed of variable domainsand constant regions comprising T cell receptors and membrane boundimmunoglobulins, any artificial binding protein displaying eitherwild-type immune effector functions or modified or artificial effectorfunctions not derivable from germline encoded heterologousimmunoglobulins or antigen receptors, and any functional fragment(s)thereof, comprising the steps of:

-   (a) genetically modifying vertebrate precursor lymphocytes, which    -   (i) are derived from primary lymphoid organs, and    -   (ii) have the potential to differentiate into mature lymphoid        lineage cells, by introducing at least one exogenous genetic        element encoding at least one binding protein or functional        fragment thereof; and-   (b) effecting differentiation of said genetically modified precursor    lymphocytes into mature lymphoid lineage cells either in vitro or in    vivo, thereby generating lymphocytes capable of producing said    binding protein or functional fragment(s) thereof, under the proviso    that the in vivo production in humans is excluded.

In order to achieve large scale production of the above binding proteinsor functional fragments thereof, it is preferred that the terminallydifferentiated lymphocytes producing the polypeptides of interest areimmortalized, or, alternatively, that the genetic information coding forthe heterologous antibodies or binding proteins are isolated andtransferred from these cells into different expression systems, wheresaid genetic information can be maintained, amplified and/or used forthe production of the respective immunoglobulins or immunoglobulin-likeproteins or functional fragments thereof.

The immortalization of said lymphocytes can preferably be achieved by:

-   (a) fusing the same to immortal myloma cells for the generation of    hybridoma cells;-   (b) infecting the same with transforming viruses, like e.g. Abelson    murine leukemia virus (A-MuLV); or-   (c) transfecting the same with an appropriate vector construct    ensuring the expression of at least one transforming oncogene, like    e.g. v-abl, or the SV40 large T antigen, that upon overexpression    leads to the immortalization of stably transfected cells;    thereby generating vertebrate lymphocytes capable of permanently    producing said binding protein or functional fragment(s) thereof.

In a preferred embodiment, the vertebrate precursor lymphocytes are ableto express at least one component of the lymphoid V(D)J recombinationmachinery and originate from jawed vertebrates comprising cartilaginousfish, bony fish, amphibians, reptilia, birds, mammals including pigs,sheep, cattle, horses and rodents including mice, rats, rabbits andguinea pigs, with murine precursor (pre) B lymphocytes from mice beingpreferred.

Primary precursor B lymphocytes in all jawed vertebrate species fromhuman down to the evolutionarily most primitive cartilaginous fish arenot only characterized by the expression of a specific set of cellsurface markers (like the combination of B220 and c-kit in mice), butalso by their potential to assemble the gene segments encoding thevariable domains of antibodies for which they express the lymphoidspecific components of the V(D)J recombination machinery, like RAG-1,RAG-2, and TdT (terminal deoxynucleotidyl transferase). Although somespecies, like birds, have different primary lymphoid organs (e.g. thebursa of Fabricius) where these precursor B lymphocytes are generated,and despite the fact that in addition to V(D)J recombination, otherspecies also use different mechanisms for antibody diversification, likesomatic hypermutation (e.g. sheep), or gene conversion (e.g. rabbits),precursor B lymphocytes from all vertebrate species are capable ofundergoing V(D)J recombination and have the potential to differentiateinto more mature B lineage cells, such that antibodies generated fromVDJ rearranged immunoglobulin gene loci can be expressed. Therefore, ifsuch vertebrate precursor B lymphocytes are isolated or generated thatare incapable of expressing endogenous antibodies, and if geneticelements encoding all or parts of heterologous immunoglobulin gene lociare stably introduced into these lymphocytes, they will gain thepotential to produce heterologous antibodies and are thus encompassed bythe present invention.

The method according to the invention makes use of vertebrate precursorB (preB) lymphocytes present in primary lymphoid organs. In the case ofmice, these cells can be isolated from fetal liver or adult bone marrow,e.g. by preparative cell sorting using and c-kit on mouse preB cells;alternatively, they can be selectively outgrown from murine fetal liveror bone marrow cell suspensions due to their strong proliferativeresponse to both bone marrow derived feeder (stromal) cells, and growthfactors such as interleukin-7 and interleukin-3.

In terms of expression of lymphoid components of the V(D)J recombinationmachinery, the potential to differentiate to mature lymphoid lineagecells and the genetic mechanism for the diversification of antigenreceptor proteins, T lineage lymphocytes are very similar to B lineagelymphocytes. It is therefore to be understood that the methods describedherein for the genetic modification of precursor B lymphocytes for thepurpose of producing heterologous antibodies or binding proteins canlikewise be applied to precursor T lymphocytes. Therefore, the presentmethods primarily described with respect to the system of murine preBlymphocytes, can also be applied to all vertebrate precursor lymphocytesfrom all jawed vertebrates as set forth before. It is to be understood,that the use of any other precursor lymphocyte system for carrying outthe principles of the present invention solely depends on thefeasibility to both prevent expression of endogenous antibodies and tointroduce heterologous genetic elements into these cells encodingheterologous antibodies or binding proteins. Since the broadapplicability of these principles will be appreciated by those skilledin the art, the general concept underlying the present invention isprimarily described with respect to the use of murine precursor B (preB)lymphocytes.

In the mouse, the development from hematopoietic stem cells to antibodysecreting plasma cells is a highly regulated process and, in vivo,depends on the ordered rearrangement of gene segments in theimmunoglobulin receptor gene loci and the regulated expression ofimmunoglobulin proteins from productively rearranged Ig gene loci (FIG.2). Usually, rearrangements occur first on both alleles of the IgH chaingene loci, with D to J_(H) gene segments rearranging first, followed byV_(H) to DJ_(H) gene rearrangements (FIG. 2). If a productiveV_(H)DJ_(H) rearrangements has occurred on one allele, μH chain can beexpressed on the cell surface as a preB cell receptor pairing withsurrogate L chain (encoded by the preB cell specific genes λ₅ andV_(preB)) (Karasuyama et al., Adv. Immunol., 63, 1-41, 1996). Theexpression of a preB cell receptor allows the differentiation of preBcells to a stage, at which V_(L) to J_(L) rearrangements are initiated,which usually occurs first on the κL chain alleles and later on the λLchain alleles (Melchers et al., Ann. Rev. Immunol., 12, p. 209-225,1994). If a productive IgL chain rearrangement has occurred, membranebound IgM and/or IgD antibodies can be expressed by so-called immature Bcells. Stimulation of peripheral, IgM/IgD positive, mature B cells withappropriate signals can lead to class switch recombination on the IgHchain alleles that changes the isotype of the expressed antibodies toany of the IgG, IgA, IgE subtypes (in mice: IgG1, IgG2a, IgG2b, IgG3,IgA, IgE, in humans: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE) (FIG. 1).

As already outlined before, the murine preB cells suitable for carryingout the invention are characterized by the expression of B220 and c-kitsurface markers, the expression of at least one of the lymphoid V(D)Jrecombination genes RAG-1 and RAG-2, and the ability to proliferate forlonger periods of time in responsiveness of stromal cells and/or IL7 orIL3 (Rolink et al., EMBO J., 10, p. 327-336, 1991; Winkler et al.,Blood, 85, p. 2045-2051, 1995). If preB cell lines and clones areestablished from wild-type mice, they usually carry DJ_(H)rearrangements on both IgH chain alleles (Alt et al., EMBO J., 3, p.1209-1219, 1984), or in some cases non-productive V_(H)DJ_(H)rearrangements (FIG. 2). However, rearrangements of their IgH chainalleles are not required to establish stromal cell, IL7 or IL3responsive cells from mice with a preB cell phenotype, as these cellscan also be established from mutant mouse strains, that are deficient inV(D)J recombination (Grawunder et al., International Immunology, 7, p.1915-25, 1995). In any circumstance, irrespective of the potential forIg gene rearrangements preB cells retain the potential to differentiateinto more mature B lineage cells in vitro, and eventually intoisotype-switched plasma cells (Rolink et al., Immunity, 5, p. 319-330,1996).

Long term proliferating murine preB cell lines and clones can beestablished from various lymphoid organs of mice, including fetal liver,fetal blood, fetal spleen, and adult bone marrow, but also other fetalor adult organs that harbour stromal cell, IL7 or IL3 responsive preBcells, especially early in life (<4 weeks of age) (Rolink et al., Blood,81, p. 2290-2300, 1993).

Mice used for the generation of the above mentioned preB cells can bewild-type mice, chimeric mice, transplanted mice or mouse strainscarrying mutations impeding V(D)J recombination on the endogenous murineIg gene loci. The above mentioned mutations may be in cis, includingdeletions of, or within the IgH diversity (D) gene segments, the IgH Jgene segments (Chen et al., Int. Immunol., 5, p. 647-656, 1993), the μHchain constant region, including the two exons for the μH transmenbraneanchor (Kitamura and Rajewsky, Nature, 356, p. 154-156, 1992), the Igκand Igλ J gene segments, the κL chain constant region (C_(κ)) (Chen etal., EMBO J., 12, p. 821-830, 1993), the λL chain constant regions(C_(λ)1-4) and the various IgH and L chain enhancers, including theheavy chain intron enhancer (EμH), the κ intron enhancer (κiE), the 3′κenhancer (3′κE), and the two λ enhancers (Eλ2-4 and Eλ3-1).

According to the invention, preB cells carrying any of the abovementioned cis-acting mutations can be used for the introduction ofexogenous genetic elements comprising part or all of heterologous,especially human immunoglobulin gene loci in non-rearranged, i.e.germline configuration. Such genetically modified preB cells can be usedfor the de novo production of antibody specificities, because the novelspecificities are only generated upon V(D)J recombination on theheterologous genetic immunoglobulin gene loci.

Alternatively, suitable preB cells can also be generated from miceincapable of rearranging endogenous immunoglobulin gene loci due totrans-acting mutations, like e.g. in the lymphoid specific recombinationactivating genes RAG-1 and RAG-2 (Mombaerts et al., Cell, 68, p.869-877, 1992; Shinkai et al., Cell, 68, p. 855-867, 1992), or in theubiquitously expressed DNA repair factors Ku70, Ku86, XRCC4, DNA ligaseIV, Artemis or the catalytic subunit of the DNA dependent protein kinase(DNA-PKcs), which are involved in the non-homologous DNA end joining,and that are also required for V(D)J recombination. For the productionof heterologous antibodies and binding proteins in preB cells carryingsuch trans-acting mutations, expression constructs are to be used thatdo not require the DNA rearrangements of V, (D) and J gene segments (seebelow).

In the case of cis-acting mutations in the mouse, these preferably needto minimally include (a) homozygous mutation(s) in the immunoglobulinheavy and the immunoglobulin κL chain gene loci. In the case oftrans-acting mutations, one of the above mentioned homozygous mutationsin a given preB cell line is sufficient to preclude the expression ofendogenous immunoglobulins being a fundamental requirement of the methodaccording to the invention.

Precursor B lymphocytes carrying cis- or trans-acting geneticmodifications interfering with the expression of endogenousimmunoglobulins can be isolated from mice carrying either naturally(e.g. scid mice) or artificially generated mutations. Alternatively,preB lymphocytes from wildtype mice can be screened for mutations ontheir immunoglobulin alleles precluding the expression of endogenousantibodies, e.g. terminally out-of-frame DJ_(H) rearrangements on bothheavy chain alleles. Mutations ablating the expression of endogenousmurine antibodies, can be introduced into hematopoietic stem cells orinto early progenitor cells, from which suitable long-term proliferatingpreB cells can be generated either upon differentiation in vitro or invivo.

Alternatively, the genetic effects referred to hereinbefore can also bedirectly introduced into at least one allele of the precursorlymphocytes to be subjected to genetic modification. The inactivation ofone allele can be sufficient for carrying out the present invention,since mutated clones can be isolated, which already carry certainmutations on the other allele (heterozygous mutations), such that ahomozygously mutated gene locus is established within the precursorlymphocyte cells to be used according to the invention.

Although it is to be understood that the invention can be realized withwild-type precursor lymphocytes, it is preferred in the method accordingto the invention, that said vertebrate precursor lymphocytes aredeprived of their potential to express endogenous antibodies and/orantigen receptors or functional fragment(s) thereof, which is achievedby isolating/selecting vertebrate precursor lymphocytes being deficientin expressing endogenous immunoglobulins or fragments thereof, and/or byintroducing into said vertebrate precursor lymphocytes at least onevector construct designed to functionally inactivate at least one alleleof at least one genetic element, which is selected from the groupconsisting of:

-   (a) the coding regions of the immunoglobulin heavy chain gene locus,    including all or parts of the V, D, and J gene segments, and any of    the coding regions for the constant region exons for μ, δ, γ, ε, and    α heavy chains, with or without their membrane spanning exons;-   (b) the coding regions of the immunoglobulin κ and/or λ light chain    gene loci, including any of the V and J gene segment coding regions,    as well as any of the constant region exons;-   (c) the coding regions of the T cell receptor α, β, γ, and δ gene    loci, including all or parts of the V, D and J gene segments, and    any of the coding regions for the α, β, γ, and δ constant region    exons;-   (d) the cis-acting immunoglobulin heavy chain gene locus enhancer    elements, including the heavy chain intron enhancer and the 3′α    enhancer;-   (e) the cis-acting immunoglobulin light chain gene locus enhancer    elements, including the κ light chain intron enhancer (κiE), the 3′κ    enhancer, and the λ2-4 and λ3-1 enhancers;-   (f) the cis-acting T cell receptor gene loci enhancer elements,    including the TCR α, β, γ, and δ enhancers;-   (g) the trans-acting recombination activating genes, RAG-1 and    RAG-2, including their promoter and enhancer elements, as well as    their coding regions; and-   (h) the trans-acting DNA repair genes essential for V(D)J    recombination, including Ku70, Ku86, the catalytic subunit of    DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV, XRCC4 and    Artemis, including their promoter and enhancer elements, as well as    the coding regions of said genes.

Preferably, the above vector constructs include gene targeting vectorscomprising regions of DNA sequence homology to said at least one geneticelement, preferably flanking a positive selection marker enablingselection of positive transfectants. For example, suitable targetingvectors contain two regions of high sequence homology (>99%), orsequence identity to the targeted gene locus, preferably flanking apositive selection marker (such as an antibiotic resistance marker),such that a double cross-over event in each of the sequence homologyregions results in targeted integration of the positive selection markerand thus in the targeted disruption of an endogenous gene (FIG. 4 a-c).Positive selection markers employed in these targeting vectors mayinclude expression cassettes conferring resistance to antibiotics likepuromycin, hygromycin B, neomycin (G418), histidinol, mycophenolic acid,zeocin and the like.

It is preferred, that said gene targeting vectors additionally comprisea pair of DNA recognition sequences for site-specific DNA recombinationenzymes, flanking the positive selection marker, enabling deletion ofsaid positive selection marker upon transfection and transientexpression of nucleic acid sequences encoding at least one of thecognate recombinase enzymes. For example, the positive selection markermay be flanked by loxP or FLP sequences recognized by thecre-recombinase or FLP recombinase enzymes, respectively (Kuhn andSchwenk, Curr. Opin. Immunol., 9, p. 183-188, 1997), allowing thesite-specific removal of the selection marker expression cassette fromthe genome of gene targeted preB cells by transient transfection of cre-or flp-recombinase expression vectors (FIG. 6 a, b). This procedureallows the repeated use of targeting vectors using the same positiveselection marker.

In a preferred embodiment, said gene targeting plasmid vectorsadditionally comprise a negative selection marker enabling selectionagainst transfectants in which said gene targeting vectors are randomlyintegrated into the genome by non-homologous recombination. For example,suitable negative selection markers selecting against random integrationof targeting constructs, may include expression cassettes for the herpessimplex virus thymidine kinase gene (HSV-TK), for the diphtheria toxingene (DT) (McCarrick et al., Transgenic Res., 2, p. 183-190, 1993), orfor its attenuated version DTtox176 (Maxwell et al., Mol. Cell. Biol.,7, p. 1576-1579, 1987). These negative selection markers are positionedin the targeting constructs, such that a targeted integration of thepositive/negative targeting construct results in the removal of thenegative selection marker (FIG. 6 a,b). Thus, only integration of thetargeting vector by homologous recombination will allow survival of thecells, and—as a consequence—selection for targeting and disruption ofpart of endogenous gene loci by homologous recombination.

It is to be understood, that the use of vertebrate precursor lymphocytesdeprived of their potential to express endogenous antibodies and/orantigen receptors or functional fragment (s) thereof is preferred.However, vertebrate precursor lymphocytes with the potential to expressantibodies and/or antigen receptors or functional fragment(s) thereofcan likewise be used according to the invention. Regardless of theactual genetic background of the precursor lymphocytes, these cells haveto be subjected to genetic modification in order to allow production ofheterologous antibodies or binding proteins. It is to be understood,that said modification will preferably be performed after appropriatetarget cells or clones have been identified by selection and/orgeneration as described before. However, it is also possible togenetically modify the cells before they are rendered incapable ofexpressing endogenous immunoglobulins or parts thereof. Furthermore, itmay even appear appropriate to simultaneously effect both steps. arerendered incapable of expressing endogenous immunoglobulins or partsthereof. Furthermore, it may even appear appropriate to simultaneouslyeffect both steps.

Accordingly, it is preferred in a further embodiment that the at leastone exogenous genetic element encoding a binding protein or (a)functional fragment(s) thereof, used for effecting the desired geneticmodification, is carried on a genetic construct selected from the groupconsisting of:

-   (a) recombinant retroviral DNA constructs comprising promoter,    enhancer and coding nucleic acid sequences operably linked to ensure    expression of, at least one binding protein or functional fragment    thereof, being either wild-type or having (a) designed mutation(s)    in the primary amino acid sequence(s) or being artificial;-   (b) recombinant plasmid-based DNA constructs comprising promoter,    enhancer and coding nucleic acid sequences operably linked to ensure    expression of at least one binding protein or functional fragment    thereof, being either wild-type or having (a) designed mutation(s)    in the primary amino acid sequence(s) or being artificial;-   (c) recombinant plasmid-based mini-immunoglobulin or T cell receptor    gene loci with unrearranged V, D and J gene segments operably linked    to allow V(D)J recombination and subsequent expression of at least    one heterologous antibody or T cell receptor, or functional fragment    thereof, being either wild-type or having (a) designed mutation(s)    in the primary amino acid sequence(s);-   (d) bacterial, yeast or vertebrate artificial chromosomes comprising    parts or all of immunoglobulin or T cell receptor gene loci in    germline configuration operably linked to allow V(D)J recombination    and subsequent expression of at least one heterologous antibody or T    cell receptor, or functional fragment thereof, being either    wild-type or having (a) designed mutation(s) in the primary amino    acid sequence(s);-   (e) bacterial, yeast or vertebrate artificial chromosomes comprising    parts or all of at least one heterologous immunoglobulin or T cell    receptor gene locus in modified arrangement designed to allow V(D)J    recombination and subsequent expression of at least one heterologous    antibody or T cell receptor, or functional fragment thereof, being    either wild-type or having (a) designed mutation(s) in the primary    amino acid sequence(s); one heterologous antibody or T cell    receptor, being wild-type with respect to the primary amino acid    sequence(s);    wherein the at least one exogenous genetic element most preferably    encodes a native or modified human antibody, a human binding    protein, a human antigen receptor, or (a) functional fragment(s)    thereof.

Furthermore, it is preferred that the at least one exogenous geneticelement encodes a heterologous or artificial receptor capable ofundergoing affinity maturation of the binding region.

For carrying out the step of genetic modification, several differentprocedures can be employed according to the invention which areexemplified in the following.

A first approach is based on the stable introduction of recombinantplasmid DNA constructs encoding parts of heterologous antigen receptorgene loci in non-rearranged, germline configuration, similar to what hasbeen achieved earlier in the context of transgenic mice. Theseheterologous antigen receptor gene loci may include V, D and J genesegments of the immunoglobulin H, κL and λL chain gene loci, as well asgene segments from any of T cell receptor (TCR) α, β, γ, or δ gene loci.

A second approach relates to the stable integration of megabase-sizedheterologous antigen receptor gene loci, including all or parts of theimmunoglobulin and/or TCR gene loci in non-rearranged, germlineconfiguration either as trans-chromosome fragments, or cloned intobacterial artificial chromosomes (BACs), yeast artificial chromosomes(YACs), or vertebrate artificial chromosomes (VACs), which has also beenperformed in the context of transgenic mice (Green et al., Nat. Genet.,7, p. 13-21, 1994; Tomizuka et al., Proc. Natl. Acad. Sci USA, 97, p.722-727, 2000).

The genetic elements of both approaches have to be stably integratedinto preB cells carrying cis-acting mutations in their endogenous murineIg gene loci, that only interfere with the rearrangement of theendogenous Ig gene loci, but that still allow the generation of diverseantibody repertoires from non-rearranged and/or germline heterologousantigen receptor gene loci. The construction of DNA constructs encodingthe human Ig gene loci relies on the knowledge of the organization ofall human Ig gene loci and their almost completely published DNAsequence as a result of the human heterologous antigen receptor geneloci. The construction of DNA constructs encoding the human Ig gene locirelies on the knowledge of the organization of all human Ig gene lociand their almost completely published DNA sequence as a result of thehuman genome project. For example, the human IgH chain gene locus islocated on chromosome 14q32.33 within a stretch of 1.25 Mbp. It contains123-129 (depending on allelic variation) V_(H) gene segments (41-47 ofthese functional), 27 D, and 6 J_(H) gene segments upstream of theconstant region gene cluster. The human IgκL chain gene locus is locatedon chromosome 2p12, comprises 1.82 Mbp, and contains 40 or 76 (dependingon allele) Vκgene segments (of which 34 or 64, respectively,functional), and 5 Jκ gene segments upstream of a single Cκ region. Thehuman IgλL chain locus is located on chromosome 22q11.2, spans 1.05 Mbp,and contains 70 or 71 Vλ (31 or 32 functional) gene segments upstream of7 to 11 tandemly repeated Jλ-Cλ gene segments.

The DNA constructs carrying part(s) or all of the germline heterologousimmunoglobulin or T cell receptor gene loci being present ontrans-chromosome fragments, or that may be cloned into BAC, YAC, or VACvector constructs, can be stably transferred into long-termproliferating target preB cells using standard gene transfer procedures,including e.g. electroporation, calcium phosphate or DEAE-dextrantransfection, liposome mediated transfer, sphero- or protoplast fusion,ballistic transfer, microinjection, or specific protein-adductformation, or a combination thereof. Stable transfectants can beselected by virtue of appropriate (drug) selection markers included inthe YAC, BAC and VAC vector backbone, comprising genes conferringresistance to e.g. puromycin, hygromycin B, neomycin (G418), histidinol,mycophenolic acid, zeocin and the like.

A third, conceptionally different approach, can be used for introducingheterologous genetic elements encoding heterologous, preferably humanantibodies and binding proteins. According to this alternative method,heterologous genetic elements are stably transferred into murine preBcells, using retroviral transduction, that has been described in thecontext of gene therapy for inherited diseases using pluripotenthematopoietic stem cells as target cells (cf. An Dong Sung et al., J.Virol. 75(8), 3547-3555 (2001)). Retroviral transfer vectors can onlyaccommodate 7-10 kb of foreign DNA, which significantly facilitates thecloning of expression vectors for heterologous, preferably humanantibodies and binding proteins. Therefore, all properties ofheterologous antibodies and binding proteins can easily and rapidly bemodified by standard molecular biology

The exon encoding the variable domains within these recombinantretroviral vectors may be monoclonal for the expression of one givenspecificity, e.g. of an existing antibody whose antigen bindingspecificity is intended to be modified by affinity maturation aftertransplantation of stably transduced preB cells into murine hosts andsubsequent immunization (FIG. 9). In addition, the exon encodingvariable domains may be derived from a library of diverse V(D)Jrearrangements. These V region libraries can be isolated fromheterologous, preferably human B lymphocytes by PCR using degenerateprimer pairs amplifying a multitude of different V_(H) gene families andJ_(H) gene segments (FIG. 9). The B lymphocytes may be derived fromindividuals that either have or have not been immunized (primed versusnaïve repertoire), or from patients suffering from autoimmune disease(autoimmune repertoire). In addition, completely synthetic libraries ofV region domains can be constructed using the in vitro assembly of Vdomain specific gene fragments that display random nucleotide sequencesin regions corresponding to the complementarity determining regions(FIG. 9).

Combinations of retroviral constructs can simultaneously or sequentiallybe transduced into preB cells allowing the expression and secretion ofcomplete heterologous immunoglobulins and dimeric binding proteins upondifferentiation of the preB cells in vitro or in vivo (FIG. 8).

Vertebrate preB cells that have been genetically modified by any of theabove mentioned methods, such that they have gained the potential toexpress heterologous antibodies or binding proteins, need to bedifferentiated into mature B lineage cells and eventually plasma cells,such that the heterologous antibodies or binding proteins can beexpressed and eventually secreted. This can either be achieved bydifferentiation in tissue culture in vitro, or upon transplantation ofthe genetically modified preB cells into appropriate vertebrate hosts invivo, under the proviso that the in vivo production in humans isexcluded.

Accordingly, it is preferred to effect the differentiation of vertebrateprecursor B lymphocytes in vitro by:

-   (a) arresting proliferation of said vertebrate precursor lymphocytes    and inducing differentiation into mature lymphocyte lineage cells by    cultivating the same in the absence of any precursor lymphocyte    growth factor; and-   (b) inducing terminal lymphocyte differentiation by further    cultivating said cells in the presence of at least one of the    following components selected from:    -   (i) soluble T cell related stimulating factors, comprising        interleukin-2, interleukin-4, interleukin-5, interleukin-6,        interleukin-10, interleukin-13, TGF-β, and IFN-γ;    -   (ii) factors activating co-stimulatory receptors of B cells,        comprising agonistic antibodies or active, recombinant ligands        specific for CD40, B7-1 (CD80), B7-2 (CD86), complement        receptors 1 (CD35) and 2(CD21), LFA-1 (CD11a), LFA-3 (CD58),        CD19, CD20, CD30, CD32, CD37, CD38, CD70, CD71, Igα (CD79α), Igβ        (CD79β), TAPA-1 (CD81), Fas (CD95), TNF-receptor1 (p55, CD120a),        TNF-receptor2 (p75, CD120b), Ox-40 (CD134), and lymphotoxin-b        receptor; and    -   (iii) B cell mitogenic factors, T cell independent antigens of        type 1, and other polyclonal activators, including        lipopolysaccharide (LPS), lipoproteins from gram negative        bacteria, polyanions, poly-dldC, pokeweed mitogen (PWM), and        anti-immunoglobulin reagents; and combinations thereof.

The in vitro differentiation in tissue culture can be achieved by a twostep process, including (a) the removal of preB cell growth factors,like e.g. IL7, or IL3, from the tissue culture medium, which will arrestproliferation of preB cells and at the same time will inducedifferentiation into cells of a mature B cell phenotype, and including(b) stimulation of the differentiated B cells by said T cell relatedand/or mitogenic, T cell independent stimuli, such that terminal plasmacell differentiation will be induced. The initial differentiation ofpreB cells into cells with a B cell phenotype by removing preB cellgrowth factors is accompanied by the induction of apoptosis. It is thusa preferred embodiment to use preB cells overexpressing anti-apoptoticgenes, like e.g. bcl-2 or bcl-x_(L). These anti-apoptotic genes can beintroduced into preB cells either by using preB cells that have beenisolated from bcl-2, or bcl-x_(L) transgenic animals, or by transfectingor transducing preB cells with overexpression vectors for any of theanti-apoptotic genes.

As an alternative to the in vitro differentiation of geneticallymodified precursor lymphocytes, these cells can also be differentiatedin vivo upon transplantation into a suitable vertebrate non-human host,resulting in migration of these cells to secondary lymphoid organs anddifferentiation into more mature lymphoid lineage cells. Preferably,said lymphocytes are co-transplanted into said host with naïve orantigen primed T helper lymphocytes, wherein the term ‘co-transplanted’is to be understood not to be limited to the simultaneoustransplantation at exactly the same time. According to another preferredembodiment, the differentiation in vivo is followed by immunization ofsaid host with at least one desired immunogenic compound or composition.In contrast to the method described herein, the transplantation ofgenetically unmodified human CD34+ hematopoietic stem cells into SCIDmice (WO 01/87058) does neither allow the generation of high affinityantibodies (because of the lack of compatible human T cells) nor anyflexibility in the type and properties of the antibodies produced bythese B cells.

Furthermore, it is preferred that said vertebrate host is a compatiblehost being deficient with respect to the generation of endogenous Bcells, T cells, and/or NK (natural killer) cells, or a combinationthereof.

As already set forth hereinbefore with respect to the selection ofappropriate sources for vertebrate precursor lymphocytes, it ispreferred that the vertebrate host is selected from jawed vertebratescomprising cartilaginous fish, bony fish, amphibians, reptilia, birds,and mammals including pigs, sheep, cattle, horses and rodents includingmice, rats, rabbits and guinea pigs, with mice being the preferred hostspecies.

The appropriate hosts serving as recipients for the transplantation ofgenetically modified vertebrate precursor lymphocytes may be wild-typewith respect to lymphocyte development, or they may preferably harbourany of various cis acting mutations inhibiting endogenous murine Ig generearrangements. These may include mutations in enhancer elements of Iggene loci, like the aforementioned E_(μ)H and κiE enhancer elements,deletions within Ig gene coding regions, like the D_(H), the J_(H) orJ_(L) gene segments and the IgC regions including their membranespanning exons. Furthermore, suitable hosts can be used carrying transacting mutations selectively impeding B (and T) lymphocyte development,e.g. mutations in lineage or lymphocyte specific transcription factorsor the RAG-1 and RAG-2 genes required for the initiation of V(D)Jrecombination (Mombaerts et al., s.a.; Shinkai et al., s.a.).

Furthermore, trans-acting mutations leading to immunodeficient hostscomprise mutations in the ubiquitously expressed DNA repair genes alsorequired for V(D)J recombination like, Ku70, Ku86, the catalytic subunitof the DNA-dependent protein kinase (DNA-PKcs), XRCC4, DNA ligase IV andArtemis. Vertebrate hosts with any of the aforementioned mutationslacking either B lymphocyte populations alone, or both B and Tlymphocytes, can then be transplanted with the genetically modified preBcells, and in the case of B- and T cell deficient mice, may beco-transplanted with histocompatible T helper cell populations. The Thelper cell populations preferably comprise naïve T helper cell,antibody primed effector T cell populations or memory T cells, or anycombination thereof. Co-transplantation of T helper cell populations mayoccur simultaneously, before or after the actual timepoint of thetransplantation of genetically modified preB cells.

Murine or vertebrate hosts whose peripheral lymphocyte populations havebeen partially or fully reconstituted by the transplantation of modifiedpreB cells and the optional co-transplantation of T helper cellpopulations can then be used for the immunization with any desiredantigen, in order to trigger an immune response to that antigen and tostimulate such lymphocytes expressing the appropriate heterologousreceptor specificity, which lead to the proliferative expansion of Bcell clones, the affinity maturation of the binding domains of theencoded heterologous antibodies or binding proteins, and the terminaldifferentiation into plasma cells secreting the heterologous antibodiesor binding proteins.

As will be apparent from the foregoing, a further aspect of the presentinvention is to provide a method for the production of any bindingprotein or functional fragment(s) thereof with the ability toselectively bind to an antigen or ligand, including any heterologousantibody, any antigen receptor composed of variable domains and constantregions comprising T cell receptors and membrane bound immunoglobulins,any artificial binding protein displaying either wild-type immuneeffector functions or modified or artificial effector functions notderivable from germline encoded heterologous immunoglobulins or antigenreceptors, and any functional fragment(s) thereof, in a manner known perse, involving the steps of:

-   (a) genetically modifying vertebrate precursor lymphocytes, which    -   (i) are derived from primary lymphoid organs, and    -   (ii) have the potential to differentiate into mature lymphoid        lineage cells, by introducing at least one exogenous genetic        element encoding at least one binding protein or functional        fragment thereof: or, alternatively, using genetically modified        vertebrate precursor lymphocytes, which    -   (i) are derived from primary lymphoid organs,    -   (ii) have the potential to differentiate into mature lymphoid        lineage cells, and    -   (iii) carry at least one exogenous genetic element encoding at        least one binding protein or functional fragment thereof:-   (b) effecting differentiation of said genetically modified precursor    lymphocytes into mature lymphoid lineage cells either in vitro or in    vivo, thereby generating genetically modified and differentiated    vertebrate lymphocytes capable of producing said binding protein or    functional fragment thereof; or, alternatively, using said    genetically modified and differentiated vertebrate lymphocytes    capable of producing said binding protein or functional fragment    thereof;-   (c) effecting expression of the binding protein or functional    fragment(s) thereof; under the proviso, that the in vivo production    in humans is excluded.

In this context, it is preferred that any of the above methods involvingthe generation of the genetically modified and differentiated vertebratelymphocytes is followed by the steps of:

-   -   (a) isolating from said differentiated lymphocytes the at least        one exogenous genetic element, and    -   (b) placing said genetic element(s) in a context enabling        production of said at least one binding protein or functional        fragment(s) thereof.

In order to achieve large scale production of the desired immunoglobulinor immunoglobulin-like protein or functional fragment thereof, thegenetic information which codes for the polypeptides of interest can beisolated from these cells and transferred into different expressionsystems, where the genetic information can be maintained, amplifiedand/or used for the production of the desired protein or part thereof.

In more detail, the open reading frames representing the coding regionsfor the heterologous antibodies or binding proteins can be isolated bystandard molecular biology methods, including PCR amplification withspecific primer pairs, and transferred into the context of differentexpression systems, allowing the continuous production of theheterologous antibodies or binding proteins. These expression systemscomprise in vitro transcription/translation systems, prokaryoticexpression systems, like e.g. E. coli, or eukaryotic expression systems,like expression in yeast cells, insect cells using baculovirusinfection, or mammalian cells.

The above-mentioned binding protein or functional fragment(s) thereofcan either display one unique specificity and can therefore bemonoclonal, or can be encoded by more than one unique specificity andcan therefore be polyclonal.

According to a preferred embodiment, said binding protein or functionalfragment(s) thereof completely or partially shares structural and/orfunctional features of a human antibody, antigen receptor, or bindingprotein, as can be assembled on the basis of the human geneticrepertoire or parts thereof.

Furthermore, it is preferred that the binding protein or functionalfragment(s) thereof to be produced is selected from the group consistingof:

-   (a) antibodies being either membrane bound or secreted, and    consisting of both heterologous heavy and light chain polypeptides    in the stoichiometric composition found in natural antibodies and    consisting of any of the known heavy (μ, δ, γ, α, ε) and/or light (κ    and λ) chain isotypes;-   (b) antibodies with combinations of heavy and light chain    polypeptides being completely human with respect to the primary    amino acid sequence;-   (c) hybrid antibodies containing heterologous heavy or light chain    polypeptides from different vertebrate species;-   (d) secreted Fab, scFv and F(ab′)₂ antibody fragments being either    completely or partially heterologous;-   (e) fragments of antibodies covalently coupled via linker peptides,    resulting in bispecific or multispecific antibody fragments;-   (f) T cell receptors of the α, β, γ, and δ isotype, antigen    receptors, and other binding proteins with structural resemblance to    proteins of the immunoglobulin superfamily;    and functional fragments thereof.

According to another aspect, the present invention provides geneticallymodified vertebrate precursor lymphocytes, which

-   (a) are derived from primary lymphoid organs and have the potential    to differentiate into mature lymphoid lineage cells,-   (b) carry at least one exogenous genetic element encoding at least    one heterologous binding protein or functional fragment thereof with    the ability to selectively bind to an antigen or ligand, including    any heterologous antibody, any heterologous antigen receptor    composed of variable domains and constant regions comprising T cell    receptors and membrane bound immunoglobulins, any heterologous    artificial binding protein displaying either wild-type immune    effector functions or modified or artificial effector functions not    derivable from germline encoded heterologous immunoglobulins or    antigen receptors, and any functional fragment(s) thereof, and-   (c) carry at least one selection marker being operably linked to    said at least one exogenous genetic element.

According to still another aspect, the present invention provides maturelymphoid lineage cells, which

-   (a) carry at least one exogenous genetic element encoding at least    one heterologous binding protein or functional fragment thereof with    the ability to selectively bind to an antigen or ligand, including    any heterologous antibody, any heterologous antigen receptor    composed of variable domains and constant regions comprising T cell    receptors and membrane bound immunoglobulins, any heterologous    artificial binding protein displaying either wild-type immune    effector functions or modified or artificial effector functions not    derivable from germline encoded heterologous immunoglobulins or    antigen receptors, and any functional fragment(s) thereof, and-   (b) carry at least one selection marker being operably linked to    said at least one exogenous genetic element.

According to a further aspect, the present invention providesimmortalized cells derived from mature lymphoid lineage cells, which

-   (a) carry at least one exogenous genetic element encoding at least    one heterologous binding protein or functional fragment thereof with    the ability to selectively bind to an antigen or ligand, including    any heterologous antibody, any heterologous antigen receptor    composed of variable domains and constant regions comprising T cell    receptors and membrane bound immunoglobulins, any heterologous    artificial binding protein displaying either wild-type immune    effector functions or modified or artificial effector functions not    derivable from germline encoded heterologous immunoglobulins or    antigen receptors, and any functional fragment(s) thereof,-   (b) carry at least one selection marker being operably linked to    said at least one exogenous genetic element, and-   (c) produce said at least one heterologous binding protein or    functional fragment thereof.

Furthermore, there are provided vector constructs suitable for at leastpartially inactivating vertebrate precursor lymphocytes to expressendogenous immunoglobulins or part(s) thereof, and genetic constructscarrying at least one exogenous genetic element encoding a heterologousantibody, an artificial binding protein, an antigen receptor, or (a)functional fragment(s) thereof, as defined hereinabove.

According to a further aspect, the present invention providespharmaceutical or diagnostic preparations, comprising at least oneantibody, artificial binding protein, antigen receptor, or functionalfragment thereof, obtained by any of the methods mentioned before,displaying either wild-type immune effector functions, or modified orartificial effector functions not derivable from germline encodedheterologous immunoglobulins or antigen receptors.

As will be appreciated, fully human or humanized antibodies orfunctional fragment(s) thereof can be used in the diagnosis, preventionand therapy of human disease by virtue of their ability to bind withtheir variable region domains to specific antigens, and, furthermore, bytheir ability to mediate immune effector functions via their constantregion domains. Some possible uses of human monoclonal antibodies, whichcan be produced according to the invention, for the diagnosis,prevention and therapy of human disease are detailed below. Fordiagnostic purposes, human monoclonal antibodies can be used for theidentification and visualization of certain pathologic conditions by theintroduction of labeled antibodies into the vascular system ofindividuals, where specific disease related antigenic structures (e.g.expressed on certain pathologic cells) can be detected. Applications inthe prevention of human disease include antibodies with the ability toblock interactions of certain cell surface receptor-ligand systems inpathologic conditions (antagonistic antibodies), or conversely, to mimicthe effect of ligand binding to cell surface receptors (agonisticantibodies) in case of the pathologic absence of a ligand. Furthermore,human monoclonal antibodies can be used for blocking the functions oftoxic substances, or of pathogens, e.g. during poisoning, inflammation,and infections. In this context, human antibodies are of particular use,because they mediate the tagging of these substances and antigens forthe removal by specialized cells of the innate immune system. Humanmonoclonal antibodies can also be used for targeting immune functions tocancerous cells by virtue of specific binding to altered self structuresexpressed on the surface of malignant cells. In case of autoimmuneconditions, human monoclonal antibodies may further be useful forneutralizing or counteracting the pathologic effects of autoimmunefactors (autoantibodies, allergic antibodies). Human monoclonalantibodies are so useful for the diagnosis, prevention and therapy ofhuman disease, because they can be introduced into the vascular systemof individuals, without causing any adverse effects, as antibodies arenormal constituents of blood plasma and fluid components of humans (e.g.mucus, saliva, lymph fluid etc.).

Deposition of Biological Material

Plasmids carrying genetic elements used in accordance to the presentinvention have been deposited under the Budapest Treaty with theDeutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) inBraunschweig, Germany, on Dec. 17, 2001, under the following AccessionNos.:

Plasmid Accession No. pBS-DT4 DSM 14703 pPGK-hygro DSM 14704 pGL2neo(m)+DSM 14705 pBS-DT4-tox176 DSM 14706

The following experimental procedures describe detailed methods for thegeneration of human monoclonal antibodies using murine preB cells thatare genetically modified in order to confer the potential for humanantibody production. Selected examples of methods are described asfollows allowing the genetic modifications of murine preB cells, suchthat endogenous antibodies can no longer be expressed, but that humanantibodies can be produced from stably delivered heterologous retroviralexpression constructs. Furthermore, experimental details are presentedrelating to the transplantation of these genetically modified murinepreB cells into appropriate murine hosts, as well as methods relating tothe immunization of these transplanted mice, the isolation of antibodysecreting cells, and their establishment as immortal monoclonal humanantibody secreting hybridoma cell lines. Although the provided methodsare complete and sufficient for the production of heterologous,especially human monoclonal antibodies using murine preB cells, that aregenetically modified, the methods should not be viewed by way oflimitation, but rather by way of illustration.

EXAMPLES Example 1 Establishment of Long-Term Proliferating, MurinePrecursor B Cells with the Potential to Differentiate into Mature BLineage Cells

Stromal cell and IL7 responsive murine precursor B cells are found infetal liver of mouse fetuses at day 15-18 post-coitum, in the bonemarrow of adult mice, as well as in the spleen of newborn animals (<3weeks of age) (Rolink et al., Blood, 81, 2290-2300, 1993). In order toisolate these preB cells, mice are sacrificed by CO₂ suffocation and therespective organs are removed under sterile conditions. Total bonemarrow cells are obtained by removing femur and tibia, opening of thebones at both ends with sterile scissors and flushing the marrow with2-5 ml phosphate buffered saline (PBS) using a 5 ml disposable syringeand a 25 or 26 gauge needle. Isolated spleen or fetal liver alsoprepared under sterile conditions is placed onto a sterile 200 meshsteel grid in a petri dish with 5-10 ml of PBS. The organs are cut intopieces and gently squashed through the metal grid with the plunger of a5 ml plastic syringe. Cell suspensions are washed once by centrifugation(200 g, 10 min, 4° C.) and resuspended in 10-20 ml PBS. All cellpreparations are carried out with ice-cold solutions, and cellsuspensions are kept on ice until transfer into tissue culture.

Establishment of individual preB cell clones is performed by limitingdilution in 96-well plates coated with semi-confluent, 3000 radγ-irradiated ST-2 (Ogawa et al., EMBO J., 7, p. 1337-1343, 1988) or PA-6(Kodama et al., J. Cell. Physiol., 118, p. 233-240, 1984) murine stromalcells grown in special stromal cell medium (see below). For limitingdilution plating, total organ cell populations can be used or,alternatively, B220⁺ or CD19⁺ surface marker positive B lineage cellsenriched by fluorescent or magnetic bead activated cell sorting (FACS orMACS) using commercial antibodies specific for these markers.

The long-term culture of murine preB cells is performed in a specialserum-free medium, containing 100 units of recombinant interleukin-7(IL-7) (see below). Individual preB cell colonies develop on stromnalcell feeders under limiting dilution conditions within 6-7 days ofculture at 37° C., in a humidified incubator under a 10% CO₂ atmosphere.Single preB cell colonies are initially transferred into 24-well plates,and then sequentially expanded into 25 cm² and eventually 75 cm² tissueculture flasks, always pre-coated with 3000 rad γ-irradiated stromalcells. After the initial expansion, preB cell densities need to be keptbetween 1×10⁵ and 2×10⁶ preB cells/ml tissue culture medium, whichrequires subculturing of the cells every two to three days.

Special serum-free tissue culture medium (Iscove and Melchers, J. Exp.Med., 147, p. 923-933, 1978) required for the establishment of long-termproliferating, stromal cell, IL-7 dependent murine preB cells(formulation for 1 liter of medium):

11 g  DMEM-powder (without bicarbonate)   10 ml  1M HEPES, pH 7.3 33.3ml  7.5% NaHCO₃ solution    8 ml  amino acid stock solution containing:  - L-alanine  600 mg   - L-asparagine  520 mg   - L-aspartate  720 mg  - L-glutamate 1800 mg   - L-proline  960 mg   - Na-pyruvate 2640 mg  + 1.6 ml Biotin/Vitamin B12 stock solution   containing      - VitaminB12 5.0 mg      - D-biotin 5.0 mg      dissolved in 20 ml + 10 μl 1M HCl  all components dissolved in 240 ml ultrapure H₂O    4 ml  cysteinestock solution containing   - L-cysteine 1.4 g   dissolved in 150 mlH₂O, 50 ml 1M HCl    5 ml  10% BSA (Bovine serum albumine) solution 0.15ml  human transferrin stock solution composed of:   - 10 ml of solutiona.) and 80 μl solution b.) a.) 1 g human transferrin dissolved in 10 mlDMEM (1.3 g/100 ml H₂O) + 100 μl 1M HEPES, pH 7.3 b.) 440 mg FeCl₃ × 6H₂O + 185 ml H₂O + 200 μl 1M HCl    5 ml  soybean-lipid stock solution  - 200 mg soybean lipids mixed with 45 ml DMEM   (1.3 g/100 ml H₂O) + 5ml 10% BSA. Sonication 3×   15 min in ice-water.   20 ml  kanamycin(5000 μg/ml) 30 μl  2-mercaptoethanol (1.43M) 10⁵ U  recombinant mouseinterleukin-7 (IL7)

All components are dissolved in a final volume of 1000 mltriple-distilled, ultrapure H₂O, filtered sterile and kept at 4° C.until use.

ST-2 or PA-6 stromal cell feeder cells need to be grown in a special,low serum containing medium as follows (formulation for 1 liter):

17.7 g IMDM-powder (without bicarbonate)    3 g NaHCO₃ 10 ml 100×non-essential amino acids (GIBCO-BRL) 10 ml 100× Penicillin/Streptomycin(GIBCO-BRL)  1 ml 5 mg/ml porcine insulin-solution  1 ml 50 mM2-mercaptoethanol  3 ml 10% primatone (ultrafiltrated to excludeproteins >10 kD) (source: Quest International, Naarden, NL) 20 ml fetalcalf serum

All components are dissolved in a final volume of 1000 mltriple-distilled, ultrapure H₂O, filtered sterile and kept at 4° C.until use.

Example 2 Stepwise Differentiation of Murine Precursor B Cells intoAntibody Secreting Plasma Cells In Vitro

Long-term proliferating, stromal cell and IL7 dependent murine preBcells retain their potential to differentiate into more mature B lineagecells (Rolink et al., s.a.), which can be achieved in vitro. Thisdifferentiation can be performed in two steps by first inducingdifferentiation to cells with an immature B cell phenotype and, second,by inducing differentiation to cells with a phenotype of antibodysecreting plasma cells.

The first differentiation stage is reached by removing IL7 from the preBcells in the continued presence of stromal cells. For this preB cellsare harvested from proliferating cultures, washed three times with serumfree preB cell culture medium lacking IL7 and plated at a density of1-2×10⁶ cells/ml onto fresh, 3000 rad γ-irradiated stromal cells in theabsence of IL7. Within a period of three days the preB cell cease toproliferate and differentiate into cells with a phenotype of immature Bcells. The phenotypic changes include e.g. the loss of c-kit, λ₅ andV_(preB) expression and the gain of CD25 and CD40 expression. In preBcells from wild-type mice, this differentiation is further accompaniedby sequential V_(H) to DJ_(H) rearrangements on the IgH chain gene locifollowed by V_(L) to J_(L) rearrangements on the IgL chain gene loci,which may result in the expression of surface bound IgM antibody on thedifferentiated B lineage cells (Rolink et al., s.a.). However, it has tobe noted that the ordered rearrangement of IgH and L gene segments isnot a prerequisite for the phenotypic differentiation of these preBcells in vitro (Grawunder et al., 1995, s.a.).

The second differentiation stage can be achieved by stimulating the IL7deprived cells with either an agonistic anti-CD40 antibody or LPS incombination with cytokines, like e.g. IL4, IL5, IL10 or TGF-β. Thistreatment will induce proliferation, class switch recombination tovarious Ig gene isotypes (depending on the cytokine combinationemployed) and eventually differentiation to antibody secreting plasmacells. In case of e.g. anti-CD40/IL4 stimulation, cells are washed threetimes in preB cell tissue culture medium, plated onto fresh,subconfluent 3000 rad γ-irradiated stromal cells in the presence of 100U/ml IL4 and 5 μg/ml anti-CD40 monoclonal antibody for 4-6 days. Large,proliferating plasma cell stage cells can be identified and separated byfluorescent activated cell sorting (FACS), based on their large sizewhich results in distinctly increased forward scatter values.

The in vitro differentiation of wild-type preB cells is usuallyaccompanied by loss of substantial amounts of cells due to growth factorwithdrawal induced apotosis. This problem can be circumvented byoverexpression of anti-apoptotic genes, like bcl-2 or bcl-x_(L) in thesecells (Rolink et al., 1993, s.a.). This can be achieved by isolatingpreB cells from mice containing a B lineage specifically expressed bcl-2transgene (Strasser et al., Curr. Top. Microbiol. Immunol., 166, p.175-181, 1990), or by transducing a bcl-2 encoding recombinantretroviral vector into the long-term proliferating, stromal cell and IL7dependent murine preB cells (see below).

Example 3 Cloning of a Retroviral Expression Construct for Murine Bcl2Overexpression

A prerequisite for the stable transduction of genes into long-termproliferating, stromal cell and IL7 dependent murine preB cells is theconstruction of a recombinant retroviral transfer vector containing anexpression cassette for a gene of interest. Retroviral transfer vectorsconsist of retroviral 5′ and 3′ long terminal repeat (LTR) sequencesflanking a retroviral packaging signal, Psi(+), a multiple cloning site(MCS) into which heterologous sequences can be inserted, and bacterialplasmid control elements required for replication and maintenance of thevector in E. coli. An example for an empty retroviral transfer vector isthe commercially available plasmid pLIB (Clontech) (FIG. 11 a).

In order to construct a retroviral expression vector containing theanti-apoptotic gene bcl-2, a strategy for coexpression of the murinebcl-2 cDNA and the hygromycinB drug selection marker is applied bylinking expression of bcl-2 and hygromycinB using an internal ribosomalentry sequence (IRES), allowing simultaneous expression of two genesfrom a single promoter. With this strategy, stable integration of thevector construct, which can be selected with the antibiotic drughygromycin B will simultaneously select cells for the expression theanti-apoptotic gene bcl-2. The generation of the retroviral vector forcoupled expression of the bcl-2 and hygromycinB cDNAs proceeds in twostages, first the assembly of the promoter-bcl-2-IRES-hygromycinBexpression cassette and, second, the cloning of this cassette into theMCS of the retroviral transfer vector pLIB (FIG. 11 a+b).

(a) Construction of the Promoter-bcl-2-IRES-hygromycinB ExpressionCassette

Both the murine bcl-2 cDNA and the open reading frame (ORF) for thehygomycinB drug resistance marker are cloned into the commerciallyavailable CMV-promoter/IRES vector pIRES (Clontech). For this, thehygromycinB ORF is amplified from plasmid pPGK-hygro (SEQ ID NO 1) bypolymerase chain reaction (PCR) using primer pair:

(P001) SEQ ID NO. 5 5′-cgTCTAGAccatgaaaaagcctgaactcaccgcgacgtctg-3′, and(P002) SEQ ID NO. 6 5′-catGCGGCCGCtattcctttgccctcggacgagtgctgggg-3′containing additional restriction enzyme recognition sites (indicated inuppercase letters) for restriction endonucleases XbaI and NotI (2-4random nucleotides 5′ of the restriction enzyme recognition sequence arealso added, because most restriction enzymes do not efficiently digestDNA, if the recognition sequence is located directly at the end of PCRamplified DNA fragments. These flanking nucleotides are indicated inlowercase letters, in order to highlight the restriction enzymerecognition sites. The sequences downstream, or 3′ of the highlightedrestriction sites correspond to DNA sequences of the fragment to beamplified. This way of presentation of primer sequences is maintainedthroughout the description of experimental examples. A PCR with primersP001 and P002 will amplify a DNA fragment of 1017 basepairs (bp)containing unique recognition sites for restriction endonucleases XbaIand NotI at the ends of the fragment. The PCR fragment is then doubledigested with XbaI and NotI restriction enzymes and is thendirectionally cloned into XbaI/NotI double digested pIRES plasmid, inorder to generate the plasmid construct pIRES-hygroB (FIG. 11 a). TheORF of the murine bcl-2a cDNA is isolated and amplified from mousespleen mRNA by reverse transcriptase coupled polymerase chain reaction(RT-PCR) using the following primers designed based on the publishedNCBI-Genbank sequences M16506 and L31532 (primer P003 binding toposition 1821-53 of M16506, and primer P004 binding to position 506-535of L31532):

(P003) SEQ ID NO. 7 5′-attGCTAGCatggcgcaagccgggagaacagggtatgataac-3′ and(P004) SEQ ID NO. 8 5′-cgcACGCGTcacttgtggcccaggtatgcacccagagtg-3containing restriction enzyme recognition sites (in uppercase letters)for NheI and MluI. A NheI/NotI double digested PCR product of 699 bysize is then directionally cloned into NheI/MluI double digested vectorpIRES-hygroB in order to generate pBcl2-IRES-hygroB.

(b) Construction of a Retroviral Bcl-2 Expressing Transfer Vector

Due to the lack of compatible restriction enzyme sites in the multiplecloning site of the empty retroviral transfer vector pLIB and the dualbcl-2/hygromycinB expression vector pBcl2-IRES-hygroB, the entireexpression cassette for Bcl-2-IRES-hygromicinB including theconstitutive CMV promoter and polyadenylation site is cloned into pLIBusing PCR with appropriated restriction enzyme recognition sites. Forthis the CMV-bcl2-IRES-hygromycinB expression cassette is PCR amplifiedwith pBcl2-IRES-hygroB as the PCR template using primers:

(P005) SEQ ID NO. 9 5′-atatGTCGACtcaatattggccattagccatattattcattg-3′ and(P006) SEQ ID NO. 10 5′-ccggATCGATccttatcggattttaccac-3′containing restriction enzyme recognition sites for SalI and ClaI (inuppercase letters). A SalI/ClaI double digested PCR product of 3714 byis then directionally cloned into the Sal/ClaI double digested pLIBvector thereby generating retroviral transfer vectorpLIB-bcl2-IRES-hygroB allowing coupled bcl-2 and hygromycinB expression.

Example 4 Retroviral Transduction of Long-Term Proliferating, StromalCell and IL7 Dependent Murine preB Cells

One of the ways to stably transfer genetic elements into murine preBcells is to use retroviral transduction (FIG. 8). For this procedure arecombinant DNA construct containing retroviral sequences necessary forpackaging of a retroviral genome (5′LTR, packaging signal Psi(+), 3′LTR)as well as heterologous sequences or genes of interest, are transientlyor stably transfected into a packaging cell line constitutivelyexpressing genes required for the production of retroviral particles.

For the transduction of stromal cell and IL7 dependent murine preBcells, the ecotropic retroviral packaging cell line GPE (Markowitz etal., J. Virol., 62, p. 1120-1124, 1988) can be used, which routinelyresults in stable transduction of preB cells at efficiencies of 50-80%.This ecotropic packaging cell line in conjunction with (recombinant)retroviral transfer vectors will only produce replication deficient(recombinant) retroviruses able to transduce murine cells. For transienttransfection of the ecotropic GPE packaging cell line, standard calciumphosphate transfection is used as detailed below:

(a) Transient Transfection of the Ecotropic Retroviral Packaging CellLine GPE:

GPE cells are grown in IMDM-based low-serum medium also used for ST-2and PA-6 stromal cells and described in example 1. One day prior totransfection, adherent GPE packaging cells are harvested bytrypsinization and seeded at 50% confluency into fresh IMDM-basedlow-serum medium and cultivated at 37° C. in 10% CO₂. The following day,20 μg retroviral DNA is transfected per T75 (75 cm²) tissue cultureflasks of GPE cells using standard calcium phosphate precipitation. 24hours post transfection, the transfected GPE cells can be used for thetransduction of recombinant retroviruses into preB cells.

(b) Retroviral Transduction of preB Cells Using Transfected GPE CellCulture Supernatant or Coculture with Transfected GPE Cells:

Two methods can be used interchangeably for the transduction ofrecombinant retroviruses produced by transfected GPE cells: The firstmethod relies on the infection of preB cells with conditioned cellculture supernatant. For this, cell culture medium from transfected GPEcells is replaced 24 hours after transfection, and is harvested afteranother 24 hours of cell culture. The recombinant retrovirus containingcell culture supernatant is then added to cultures of proliferating preBcells at ratios of 1:8 (vol) to 1:1 (vol) in the presence of 8 μg/mlpolybrene and incubation is continued for 3-6 hours at 37° C. Cells arethen harvested, the polybrene/retrovirus containing medium is removed,and cells are plated at a density of 1-2×10⁵ onto fresh, 3000 radγ-irradiated stromal cells in preB cell medium containing 100 U rIL7.Retrovirally transduced preB cells are then harvested 24 hours later.

The second method relies on the coculture of preB cells withtransfected, recombinant retrovirus producing GPE cells. For this,log-phase preB cells are harvested and resuspended in fresh preB cellculture medium containing 100 U rIL7 and 8 μg/ml polybrene at a densityof 2-4×10⁵ cells and are plated onto transfected GPE cells 24 hours posttransfection with retroviral constructs. PreB cells are co-cultured for6 hours at 37° C., they are then harvested and plated onto fresh, 3000rad γ-irradiated stromal cells in preB cell medium containing 1000 UrIL7 for recovery. Retrovirally transduced preB cells are then harvestedand can be utilized 24 hours later.

Example 5 Inactivation of Endogenous Immunoglobulin Heavy and LightChain Gene Loci in Long-Term Proliferating, Murine Precursor B Cells

The first step for the generation of heterologous, preferably humanantibodies from murine precursor B cells, is the inactivation of theendogenous, murine IgH and IgL, which can, among other methods, e.g. beaccomplished by gene targeting within the immunoglobulin gene locus inpreB cells. In theory, it would be required to inactivate all threemurine immunoglobulin gene loci, i.e. the IgH, IgκL and IgλL chain geneloci. However, the murine λL chain gene locus comprises only threefunctional V-J-C clusters with one gene segment each and only verylimited diversity is comprised in λL chains. Furthermore, less than 5%of murine B cells express λL chain gene loci. Therefore, in practicalterms, it is sufficient to inactivate the endogenous, murine IgH and κLchain gene loci in order to assure that upon transfer of heterologousIgH and IgkL chain genes or gene loci, the vast majority of B cells(>95%) produced from preB cells will express heterologous antibodies.

(a) Isolation of preB Cells with Naturally Occurring IrreversiblyNon-Functional IgH Alleles.

Generally, the most controlled way for inactivation of the endogenousIgH and κL chain gene loci is to use gene targeting (see below).However, in case of the IgH chain gene locus, another method can beapplied:

Long-term proliferating, stromal cell IL7 dependent murine preB cellscarry DJ_(H) rearrangements on both of their heavy chain alleles. Due toimprecision in the D to J_(H) joining process, these DJ_(H)rearrangements may occur such that D gene segments can be read in allthree possible reading frames relative to the fixed reading frame of theJ_(H) gene segment. These reading frames have arbitrarily beendesignated reading frames I, II and III (Kaartinen and Mäkelä, Immunol.Today, 6, p. 324-330, 1985). Most D gene segments contain stop codons,if a DJ_(H) junction occurs in reading frame III (Kaartinen and Mäkelä,s.a.), such that a functional H chain can never be expressed from such aheavy chain allele.

Therefore, statistically roughly 1/9 (11.1%) of all stromal cell, IL7dependent preB cells carry DJ_(H) rearrangements in reading frame IIIwith stop coding on both of their IgH chain alleles. These preB cellscan be isolated by subcloning under limiting dilution conditions andscreening for the desired non-functional rearrangements. To ensure theisolation of stable clones, rearrangements need to be screened for twonon-functional DJ_(H)4 gene rearrangements, which cannot be reverted bysecondary rearrangements because the last J_(H) gene segment has beenused up. Such clones can be used for further targeted inactivation ofthe κL chain alleles.

(b) Generation of Targeting Constructs for the Murine IgH and IgκL ChainLoci

Two examples for methods resulting in gene targeted inactivation of boththe endogenous IgH and IgκL chain gene loci are presented as examplesfor many cis-acting mutations that would similarly lead to preB cellswithout the potential to express endogenous IgH and IgκL chain proteins(FIG. 5 a,b). For both targeting strategies, first, emptypositive-negative gene targeting vectors need to be constructedcontaining unique cloning sites allowing the insertion of DNA sequenceshomologous to the target gene locus, a positive drug selection marker,like neomycin or puromycin, flanked by loxP sites and a negativeselection marker, like diphtheria toxin, or herpes simplex virusthymidine kinase (HSV-tk), which will select against random integrationof the gene targeting vector into the genome of cells. The presence of aloxP flanked (floxed) positive selection marker will allow thesequential gene targeting of both alleles in the same cell clone usingthe same antibiotic, because, after successful targeting of the firstallele, the antibiotic resistance marker can be deleted from cells upontransient expression of cre recombinase deleting any nucleotide sequencelocated in between the cognate loxP recognition sites. The emptypositive-negative targeting vectors are constructed as follows (FIG. 6a,b):

Step 1: Extending the Multiple Cloning Site of pBluescript SK(+)

Two complementary synthetic DNA oligomers:

(P007) SEQ ID NO. 11 5′-TCGAccggtacctaggcgcgccatcgatatcgctagctcgagctcagatctGTAC-3′ and (P008) SEQ ID NO. 125′-agatctgagctcgagctagcgatatcgatggcgcgcctaggtaccg g-3′are annealed by mixing 100 pmol of each oligomer in 50 μl of 50 mMTris/HCl, 100 mM NaCl, pH8.0. The mixture is then denatured for 2minutes at 95° C. and slowly cooled from 65° C. to room temperaturewithin 20 min. This will generate the following double stranded DNAlinker with XhoI and KpnI compatible overhangs (indicated in uppercaseletters):

The annealed synthetic DNA linker is then ligated into XhoI-KpnIlinearized commercially available pBluescriptSK(+) (Stratagene)resulting in plasmid pBSL containing an extended multiple cloning site(MCS+, see FIG. 6 a) required for the following cloning steps.

Step 2: Insertion of Expression Cassettes for Wild-Type or AttenuatedDiphtheria Toxin α as a Negative Selection Marker Against RandomIntegration of Targeting Constructs

As a negative selection marker that allows selection against randomchromosomal integration of gene targeting constructs, an expressioncassette for diphtheria toxin α (McCarrick et al., s.a.), or itsattenuated version, diphtheria toxin α (DT-αtox176) (Maxwell et al.,s.a.), under control of a constitutive β-actin promoter can be used.Although other negative selection markers, like the herpes simlex virusthymidine kinase gene (HSV-tk), is commonly used in gene targetingexperiments with mouse embryonic stem cells, diphtheria toxin α and itsattenuated mutant tox176 have been proven to be effective for genetargeting in somatic cells (Grawunder et al., Mol. Cell, 2, p. 477-484,1998). The diphtheria toxin expression cassettes are isolated as 2.1 kbBglII-XbaI fragments from plasmids pBS-DT4 (SEQ ID NO. 2), orpBS-DT4tox176 (SEQ ID NO. 3; see FIG. 6 a) and are ligated intoBglII-NheI linearized pBSL resulting in plasmids pBSL-DT4 andpBSLDT4tox176, respectively (see FIG. 6 a).

Step 3: Insertion of loxP Flanked Positive Drug Selection Markers intopBSL-DT4 and pBSLDT4tox176 for the Generation of Empty Gene TargetingVectors

The next step for the generation of positive/negative gene targetingvectors is the insertion of a positive drug selection marker into thepBSL-DT4 or pBSL-DT4-tox176 vector, upstream of the diphtheriaexpression cassette. As representative examples the insertion of loxPsite flanked neomycin, puromycin and hygromycin B expression cassettesupstream of the diphtheria toxin α expression cassette in pBSL-DT4 arepresented. However, it should be noted that identical cloning strategiescan be applied to the pBSL-DT4tox176 vector, containing the attenuatedversion of diphtheria toxin α. Furthermore, other positive drugselection markers conferring resistance e.g. to histidinol, mycophenolicacid, bleomycin, or zeocin may be used.

In each case, the positive antibiotic selection marker cassette will beflanked by loxP sites, that can be recognized by the cre recombinaseenzyme, and which can be used to delete the drug selection markerlocated between the loxP sites upon transient transfection of a crerecombinase expression vector, for sequential use of the same selectionmarker.

i) Insertion of a Floxed Neomycin Resistance Cassette:

A loxP site flanked neomycin resistance cassette can be isolated as a1497 bp Bsp120I-XbaI digested DNA fragment from plasmid pGL2neo(m)+ (SEQID NO. 4) and can be ligated into NotI-XbaI linearized pBSL-DT4 togenerate the empty targeting vector pBSL-flneo-DT4.

ii) Cloning of Floxed Puromycin and HygromycinB Cassettes (FIG. 6 b):

Because floxed antibiotic selection markers other than neomycin arerarely found, an essential step for the insertion of loxP flankedpuromycin or hygromycinB markers into pBSL-DT4 is the preparatory stepof inserting two loxP sites. This can be achieved by inserting intopBSL-DT4 annealed synthetic DNA oligomers. The synthetic loxP sites arecontained in the following two complementary DNA oligos:

(P009) SEQ ID NO. 13 5′-CTAGATAACTTCGTATAGCATACATTATACGAAGTTATACGCGTATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ and (P010) SEQ ID NO. 145′-CTAGATAACTTCGTATAATGTATGCTATACGAAGTTATACGCGTATAACTTCGTATAATGTATGCTATACGAAGTTAT-3′

These two DNA oligomers are annealed as described in example 5(b), step1, resulting in a double stranded DNA fragment containing two loxP sitesseparated by a unique MluI restriction site. In addition, the annealedsynthetic DNA oligomers contain two 5′-CTAG single-stranded overhangs,which can directly be ligated into compatible restriction site overhangs(generated e.g. by SpeI, NheI, XbaI, or AvrlI restriction enzymes).

The annealed double loxP site containing DNA fragment is then ligatedinto SpeI linearized pBSL-DT4 resulting in vector pBSL-DT4-2loxP (FIG. 6b).

Expression cassettes for puromycin or hygromycinB resistance can be PCRamplified using plasmids pPur (Clontech) or pPGK-hygro (SEQ ID NO. 1) asPCR templates, respectively. The primers used for PCR amplification ofthe 1366 by puromycin expression cassette from pPur (containing thepuromycin ORF under control of the simian virus40 early promoter) are:

(P011) SEQ ID NO. 15 5′-tcgACGCGTggaatgtgtgtcagttagggtgtggaaag-3′ and(P012) SEQ ID NO. 16 5′-tcgACGCGTttggacaaaccacaactagaatgcagtg-3′

Both primers contain recognition sites for the restriction endonucleaseMluI (indicated in uppercase letters), such that the resulting PCRproduct can be cloned using this restriction enzyme.

After MluI digestion of the isolated PCR product the puromycin markerfragment is ligated into MluI linearized pBSL-DT4-2loxP generating theempty targeting vector pBSL-flpur-DT4 (FIG. 6 b).

The primers used for PCR amplification of the 2007 by hygromycin Bexpression cassette from pPGK-hygro (SEQ ID NO. 1; containing thehygromycinB ORF under control of the phospho glycerat kinase promoter)are:

(P013) SEQ ID NO. 17 5′-tcgACGCGTgaattctaccgggtaggggaggcgcttttc-3′ and(P014) SEQ ID NO. 18 5′-tcgACGCGTggaattagaacttggcaaaacaatactgag-3′

Both primers contain recognition sites for the restriction endonucleaseMluI (indicated in uppercase letters), such that the resulting PCRproduct can be digested and ligated using this restriction enzyme.

After MluI digestion of the isolated PCR product the hygromycinB markerfragment is ligated into MluI linearized pBSL-DT4-2loxP generating theempty targeting vector pBSL-flhyg-DT4 (FIG. 6 b).

Step 4: Cloning of Final Targeting Vectors for Murine IgH and κL ChainGene Loci, as well as for the RAG1 Gene

As representative examples for cloning of targeting vectors forendogenous gene loci, the cloning strategies for gene targeting vectorsuseful for the cis-acting targeted mutation of the endogenous IgH andIgκL chain gene loci, and for the trans-acting mutation of the RAG1 genein murine preB cells are described.

i) Construction of IgH Chain Targeting Vectors (FIG. 5 a)

Inactivation of the endogenous murine IgH chain gene locus in preB cellscan be achieved by generating many different cis-acting targeteddeletions on both IgH chain alleles. One of IgH inactivating deletion isfor instance the deletion of all D_(H) and J_(H) gene segments, which isdescribed here as one of the possibilities to render the endogenous IgHalleles incapable of producing endogenous immunoglobulins. Targetingvector construction is presented here using a “floxed” neomycintargeting construct as an example (see FIG. 5 a). For the cloning ofgene targeting vectors containing other positive antibiotic selectionmarker, like e.g. puromycin or hygromycinB , similar strategies may beapplied.

Stromal cell, IL7 dependent murine preB cells usually carry DJ_(H)rearrangements on both alleles (see above). This fact needs to be takeninto account for the design of targeting vectors. Regardless of the typeof DJ_(H) rearrangement, the DNA sequences intervening the variable genesegments and the D gene segment cluster, as well as the interveningsequences between the J_(H) gene cluster and the C_(μ) coding region arestill present on both IgH alleles in all preB cells. Targeting vectorsfor DJ_(H) rearranged alleles therefore have to contain homology regionsupstream of the most 5′ located D element (D_(FL16.1)) and downstream ofthe most 3′ located J_(H) gene segment (J_(H)4).

The genomic organization of one possible D_(FL16.1)-J_(H4) rearrangedallele with surrounding genomic DNA sequences is depicted in FIG. 5 a.As a general strategy for gene targeting, a short region of DNA sequencehomology (1-1.5 kb) located upstream of the region or gene to be deletedis cloned into a unique restriction enzyme recognition site upstream ofa “floxed” positive selection marker (here NotI), and a longer region ofDNA sequence homology (>2.5 kb) located downstream of the region or geneto be deleted is cloned into a unique restriction enzyme recognitionsite located between the “floxed” positive selection marker and thediphtheria toxin α expression cassette (FIG. 5 a) (here AscI).Expression of the negative selection marker (diphtheria toxin α) istherefore only prevented, if homologous recombination occurs in the longregion of DNA sequence homology of the targeting vector and theendogenous gene locus, such that the DT-α expression cassette will belost upon targeted integration of the targeting vector into thechromosomal DNA. If homologous recombination further occurs in the shorthomology arm, the region located between the double crossover will bereplaced by the positive drug selection marker. Thus using antibioticselection on preB cells transfected with the targeting construct resultsin strong selection for the targeted integration of the targeting vectorby homologous recombination resulting in the deletion of the endogenousgene locus.

A short region of homology upstream of the most 5′ located D element(D_(FL16.1)) can be PCR amplified from mouse genomic DNA using thefollowing primers designed based on the published NCBI-Genbank sequenceAF018146:

(P015) SEQ ID NO. 19 5′-aatGCGGCCGCgaacctcctgtgttgcaagcacaaatggg-3′, and(P016) SEQ ID NO. 20 5′-aatGCGGCCGaggcagcacggttgagtttcagttgtcatc-3′

Nucleotides of the appended NotI restriction enzyme recognition sitesintroduced for cloning purposes are indicated in uppercase letters. Theprimers will amplify a 1465 bp genomic PCR fragment that can be digestedwith NotI restriction enzyme and ligated into NotI linearizedpBSL-flneo-DT4 generating pBSL-5′J_(H)-flneo-DT4 (FIG. 5 a). The longregion of sequence homology downstream of the J_(H) gene segmentcluster, also comprising the EμH heavy chain intron enhancer, can be PCRamplified from mouse genomic DNA using the following primers designedbased on published NCBI-Genbank sequence file J00440 (nucleotides of theappended AscI restriction enzyme recognition site are indicated inuppercase letters):

(P017) SEQ ID NO. 21 5′-ttGGCGCGCCgtaagaatggcctctccaggtctttatttt-3′ and(P018) SEQ ID NO. 22 5′-ttGGCGCGCCagctcagctcagctcaccccagctcagctc-3′

The primers will PCR amplify a 3215 bp genomic PCR fragment from mousegenomic DNA that can be digested with AscI restriction enzyme andligated into AscI linearized pBSL-5′J_(H)-flneo-DT4 generating the finalJ_(R) targeting vector pBSL-5′J_(H)-flneo-3′J_(H)-DT4 that can be usedto delete all remaining D_(R) and J_(H) gene segments in DJ_(H)rearranged preB cells (FIG. 5 a).

ii) Construction of κL Chain Targeting Vectors (FIG. 5 b)

Like in the case of the IgH chain locus, the κL chain gene loci may beinactivated by several cis-acting mutations, including e.g. deletions ofall of the J_(κ) gene segments, the κL chain intron enhancer (κiE), theconstant κL chain region (C_(κ)), or the 3′κ enhancer (3′κE). As anexample, targeting constructs and methods are described here that can beused for the targeted deletion of all of the J_(κ) gene segments.Furthermore, construction of targeting constructs described herein ispresented only for targeting constructs containing a “floxed” neomycinresistance marker. Cloning of targeting constructs containing “floxed”puromycin or hygromycinB expression cassettes are performed in analogousfashion. A short region of homology upstream of the J_(κ) gene segmentcluster is PCR amplified from mouse genomic DNA using the followingprimers designed according to published NCBI-Genbank entry V00777(nucleotides of the appended NotI restriction enzyme recognition siteused for cloning purposes are printed in uppercase letters):

(P019) SEQ ID NO. 23 5′-tatGCGGCCGcttatctttctcctttattaacggttgctg-3′ and(P020) SEQ ID NO. 24 5′-tatGCGGCCGCacagtggtagtactccactgtctggctg-3′

Primer P019 binds to position 1-30 and primer P020 to position 711-738of NCBI-Genbank entry V00777. The resulting 738 by PCR fragment isdigested with NotI restriction enzyme and is ligated into NotIlinearized empty targeting vector pBSL-flneo-DT4 generatingpBSL-5′J,-flneo-DT4 (FIG. 5 b). The long region of DNA sequence homologydownstream of the J_(κ) gene segment cluster comprising the κiE and theconstant kL chain region (Ck) is PCR amplified from mouse genomic DNAusing the following primers designed based on published NCBI-Genbankentry V00777 (nucleotides of the appended AscI restriction enzymerecognition site are printed in uppercase letters):

(P021) SEQ ID NO. 25 5′-ttGGCGCGCCtgtgtaagacacaggttttcatgttaggag-3′ and(P022) SEQ ID NO. 26 5′-ttGGCGCGCCtgcttcgccaagtttactgggtaggttg-3′

Primer P021 will bind to position 2129-2158, and primer P022 to position5456-5483 of NCBI-Genbank sequence V00777. The resulting 3379 by PCRfragment is digested with AscI restriction enzyme and is ligated intoAscI linearized vector pBSL-5′J_(κ)-flneo-DT4 generatingpBSL-5′J_(κ)-flneo-3′J_(κ)-DT4 (FIG. 5 b).

iii) Generation of a RAG Locus Targeting Vector (FIG. 5 c):

As an alternative to inactivating the potential of preB cell to expressendogenous immunoglobulins by introducing cis-acting mutations withinthe IgH and IgκL chain gene loci, preB cells can also be renderedincapable for rearrangement of their endogenous Ig genes, by introducingtrans-acting mutations. This can for instance be achieved by targetingone of the two recombination activating (RAG) genes that are essentialfor Ig gene rearrangements. Murine preB cells with deletions in eitherthe RAG-1, or the RAG-2 gene will be unable to rearrange immunoglobulingene segments and are therefore unable to express endogenousimmunoglobulins.

As an example, the construction of a targeting vector is describedallowing the complete deletion of the ORF of the RAG-1 gene. A short armof DNA sequence homology of about 1 kb of genomic sequence upstream ofthe RAG-1 coding region is amplified by the following primer pairdesigned based on published NCBI-Genbank sequence AC084753:

(P023) SEQ ID NO. 27 5′-tataGCGGCCGcttctgctcctcttctttagtactggattc-3′ and(P024) SEQ ID NO. 28 5′-tataGCGGCCGCgttggctaagctacctgggaacaatggggg-3′

NotI cloning sites appended to the 5′ end of each primer are highlightedin uppercase letters and are used for the cloning of a 1095 bp PCRfragment containing the genomic region upstream of the RAG-1 openreading frame (FIG. 5 c). The PCR product is digested with NotI andligated into the unique NotI site of a positive-negative targetingvector, e.g. pBSL-flneo-DT4. A plasmid clone containing the short arm ofhomology upstream of the RAG-1 coding region in correct orientation isisolated and designated pBSL-5′R1-flneo-DT4.

For the cloning of the long arm of a targeting construct for the RAG-1gene, a genomic region of approximately 3 kb directly downstream of theRAG-1 coding region (FIG. 5 c) is amplified by the following primerpair, designed based on Genbank sequence AC084753:

(P025) SEQ ID NO. 29 5′-ttGGCGCGCCataggatctccacatagaagttggtatttgcc-3′and (P026) SEQ ID NO. 30 5′-ttGGCGCGCCgtgggcacacatatgtcatcccagttaccc-3′

Recognition sequences for the AscI restriction enzyme are appended tothe 5′ end of each primer and are used for the cloning of the 2954 bylong PCR fragment containing the long arm of DNA sequence homogydownstream of the RAG-1 open reading frame. After AscI digestion, thisPCR product is cloned into the unique AscI restriction site of thevector pBSL-5′R1-flneo-DT4. A plasmid clone containing the long arm ofhomology downstream of the RAG-1 coding region in the correctorientation is isolated and designated as pBSL-5′R1-flneo-3′R1-DT4 (FIG.5 c) and can be directly used for the targeted deletion of theendogenous RAG-1 gene in preB cells.

Step 5: Targeting of Endogenous Gene Loci in Long-Term Proliferating,Stromal Cell, IL7 Dependent Murine preB Cells

For the sequential targeted deletion of both alleles of an endogenousgene locus, two different strategies can be applied. One strategy is toutilize targeting vectors with two different positive selection markersfor the targeting of the two different alleles. Alternatively, anotherstrategy is to use the same targeting construct twice for the sequentialtargeting of both endogenous alleles. However, in case of the latterstrategy targeting of the first allele has to be followed by thetransient expression of an expression vector for cre-recombinase intopreB cells, which will lead to the permanent deletion of the loxP siteflanked drug selection marker, such that the same antibiotic drugselection may be used for the second targeting of the other allele.

As an example, the experimental procedure for the sequential targetingof both alleles of an endogenous gene according to the first strategy,i.e. using targeting vectors with two different antibiotic resistancemarkers (puromycin and hygromycinB) is described.

For targeting of a gene locus on the first allele, 20 μg of linearizedtargeting vector with a puromycin resistance marker is transfected into10⁷ preB cells resuspended in PBS by electroporation at 350V with 960 μFcapacitance using a BioRad electroporator and a 0.4 cm electroporatorcuvette. The cells are then plated in IL7 growth medium at a density of5×10⁴ cells/well into 96well plate cultures coated with a subconfluentlayer of 3000 rad irradiated puromycin resistant ST-2 stromal cells. 30hours post transfection puromycin is added at a final concentration of 2μg/ml. Puromycin resistant colonies are isolated 7-10 days posttransfection and expanded on fresh, irradiated and puromycin resistantST-2 stromal cells under continued selection with 2 μg/ml puromycin.Individual puromycin resistant preB cell clones are analyzed for thetargeted integration of the targeting vector into one allele by standardPCR and genomic southern blot hybridization analysis. PreB cell cloneswith a targeted integration of the targeting vector on one allele areagain transfected, this time with 20 μg of linearized targeting vectorcontaining a hygromycinB resistance marker under the same conditions asdescribed above. Transfected preB cells are then plated in IL7 growthmedium at a density of 5×10⁴ cells/well into 96well plate culturescoated with a subconfluent layer of 3000 rad irradiated puromycin andhygromycinB double resistant ST-2 stromal cells. Double resistant preBcell clones are selected 30 hours post transfection by addition of 2μg/ml puromycin and 400 μg/ml hygromycinB. Puromycin and hygromycin Bdouble resistant colonies are isolated 7-10 days post transfection andexpanded on fresh, irradiated and puromycin/hygromycinB double resistantST-2 stromal cells under continued selection with 2 μg/ml puromycin and400 μg/ml hygromycinB. Individual puromycin/hygromycinB double resistantpreB cell clones are analyzed for the targeted integration of thetargeting vector into the second allele again by standard genomic PCRand/or southern blot analysis.

Puromycin/hygromycinB double resistant preB cell clones with twotargeted alleles may then transiently be transfected with a constitutivecre-recombinase expression vector in order to repeat gene targetingexperiments on other gene loci and alleles. This can be done bytransiently transfecting preB cells with 20 μg of supercoiledcre-recombinase expression vector by electroporation as described above.The transiently transfected cells are then plated under limitingdilution conditions in IL-7 growth medium into several 96 well platesonto 3000 rad irradiated, subconfluent ST-2 stromal cells. After 7 daysof culture a set of 48 clones is replica plated onto normal ST-2 stromalcells for continued culture, and onto either puromycin resistant orhygromycinB single resistant ST-2 cells and the respective selection.Previously puromycin/hygromycinB double resistant preB cell clones thatbecame sensitive two both antibiotic drugs have likely undergonecre-recombinase mediated deletion of both loxP flanked resistancemarkers. Once the cre-recombinase mediated deletion of the tworesistance markers has been verified by southern blot analyses, theclones can be subjected to the additional gene targeting procedures forother endogenous gene loci and alleles.

Example 6 Construction of Retroviral Expression Constructs for HumanImmuno-Globulin Polypeptides

In order to re-program murine stromal cell, IL7 dependent preB cells forthe production of human immunoglobulins, novel retroviral expressionvectors encoding human antibody polypeptides can be used. Methods forthe generation of recombinant retroviral vectors are described thatallow the regulated expression of human IgH and L chain proteins,depending on the B cell differentiation stage, first as membrane-boundand later as secreted immunoglobulins. It should be noted that any typeof heterologous binding protein can be cloned into these retroviralvectors allowing the expression of any type of heterologous and humanbinding protein. The description of the cloning of retroviral transfervectors for the expression of human IgH and IgL chains described herein,should therefore only be viewed by way of illustration and not by way ofexclusion. Recombinant retroviral vectors can accommodate up 7-10 kb ofheterologous DNA sequences, which requires the design of separateconstructs for IgH and L chain glycoproteins including all the requiredcell-type and differentiation stage specific promoter, enhancer andcontrol elements, as well as endogenous Ig gene splice signals. Thesecontrol elements are necessary to ensure proper expression, membranedeposition and secretion of immunoglobulins, their affinity maturation,in the various B cell differentiation stages, as well as high levelsecretion of heterologous antibodies in plasma cells. Individualretroviral constructs for IgH and L chains can be co-transduced intopreB cells upon which the constructs stably integrate into the genome ofthe infected preB cells. If murine preB cells are used which aredeprived of the potential express endogenous immunoglobulins (seedescription above), then only heterologous or human antibodies orbinding proteins will be expressed in B lineage cells upondifferentiation of the retrovirally transduced preB cells.

(a) Generation of Retroviral Constructs for the Regulated Expression ofIgH Chains

Because the retroviral constructs for the various IgH and L chainproteins will eventually be transduced into murine cells, the promoterand enhancer elements controlling Ig expression will be from murineorigin in order to ascertain optimal interactions with B lineagespecific murine transcription factors.

The starting vector from which the retroviral expression constructs aregenerated is the empty retroviral cloning vector pLIB (Clontech) (FIG.12 a). The generation of a recombinant retroviral expression vector forhuman immunoglobulin heavy chains is a multistep cloning process, whichinvolves the sequential cloning of promoter and enhancer elements, aswell as the coding regions for the various variable and constant regionsof human antibodies into the empty retroviral cloning vector pLIB. In afirst step, a murine V_(H) promoter is PCR cloned into the unique ClaIrestriction site of the multiple cloning site of pLIB. This is done byPCR amplification of a mouse V_(H) promoter element from mouse genomicDNA using the following primers designed based on the publishedNCBI-Genbank database accession number X71119.

(P027) SEQ ID NO. 31 5′-ccATCGATtgactggatgcttgttaattctaataag-3′, and(P028) SEQ ID NO. 32 5′-ccATCGATGGATCCtgtgtgccagtaactgtagagagaac-3′

These primers will PCR amplify a 394 bp V_(H) promoter fragment whichwill contain restriction enzyme recognition sites for ClaI (ATCGAT) attheir 5′ and 3′ ends, because they are incorporated at the 5′ ends ofprimers P027 and P028. In addition to ClaI sites in both primers, thereverse primer P028 contains an additional BamHI restriction site(GGATCC, underlined) introducing another unique restriction site intothe PCR product (see FIG. 12 a), which will be required for furthercloning steps. After ClaI restriction enzyme digestion of the PCRfragment, the amplified V_(H) promoter PCR fragment including the novelBamHI site is ligated into ClaI linearized pLIB vector. Ligationproducts containing the V_(H) promoter in the desired orientation(opposite to the transcriptional orientation of the 5′LTR promoter) areidentified by diagnostic restriction enzyme digestions and DNAsequencing and are designated pAB-1 (FIG. 12 a).

The second step is the cloning of the murine intron heavy chain enhancer(EμH) with its flanking matrix attachment regions (MARs) into retroviralvector pAB-1. The EμH enhancer element with its flanking (MARs) is knownto be required for the efficient expression of immunoglobulin proteinsin B lineage cells. The cloning of the EμH enhancer element is performedby amplifying a 983 bp PCR fragment from mouse genomic DNA using thefollowing primers designed according to published NCBI-Genbank sequenceJ00440:

(P029) SEQ ID NO. 33 5′-cgGGATCCAAGCTTagagaggtctggtggagcctgcaaaagtcc-3′and (P030) SEQ ID NO. 34 5′-gatcGCGGCCGCtctagataattgcattcatttaaaaaaaaaatatttc-3′

Restriction sites (BamHI/HindIII for P029 and NotI for P030) appended tothe 5′ end each primer are highlighted in uppercase letters. The PCRproduct can then be digested with BamHI and NotI restriction enzymes andis directionally ligated into BamHI/NotI linearized retroviral vectorpAB1, resulting in vector pAB-2. The sense primer P029 contains thesequence for an additional HindIII site (underlined), which will giverise to a unique restriction enzyme recognition site in pAB-2, requiredfor later cloning steps.

Next, a DNA fragment containing the murine 3′α enhancer is added to theconstruct pAB-2. The 3′α enhancer is known to be required for optimaland high level expression and secretion of antibodies at thedifferentiation stage of plasma cells. The 3′ a enhancer is known tocontain four DNAsel hypersensitivity sites (HS1, HS2, HS3, and HS4) thatcomprises various binding sites for late B cell/plasma cell specifictranscription factors. In reporter gene assays, it was shown that HS1and 2, which are located within a 0.6 kb genomic DNA fragment, confer atleast 80% of the 3′α enhancer activity with little contribution of HS3and 4 that are highly homologous in DNA sequence.

All four 3′α enhancer components, HS1, 2, 3 and 4, constitute aso-called locus control region (LCR), which is able to regulate thetranscriptional activity of a given gene locus independently of flankingDNA sequences. A basic retroviral expression vector for immunoglobulinheavy chains should therefore at least contain the HS1,2 regions of the3′α enhancer which can be PCR amplified from mouse genomic DNA usingprimers designed according to the published NCBI-Genbank sequence entryX96607 (recognition sequences for additional restriction enzyme sitesrequired for the cloning of the PCR fragment or later cloning steps arehighlighted in uppercase letters):

(P031) SEQ ID NO. 35 5′-gcatGTCGACACGCGTgggggctcagatatcagtaccagaaacaagg-3′, and (P032) SEQ ID NO. 365′-cgatGTCGACCTCGAGttggagtcacaggcctgtctccatgtgg-3′

In addition to 5′ terminal SalI sites (GTCGAC) in each of the primers,the sense primer P031 contains an additional MluI site (underlined) andthe reverse primer P028 contains an additional XhoI site (underlined).Primers P031 and P032 will amplify a 644 bp PCR fragment that can bedigested with SalI restriction enzyme and ligated into SalI linearizedvector pAB-2. A clone with the insert in the desired orientation (seeFIG. 12 b) is determined by diagnostic restriction enzyme digestion andDNA sequencing, and is designated pAB-3 (FIG. 12 b).

Optionally, the further 3′α enhancer components HS3 and 4 can beincluded in the retroviral vector constructs for IgH expression. Forthis a 466 by fragment comprising the 3′α enhancer HS4 region can be PCRamplified from mouse genomic DNA as a template using the followingprimers designed according to published NCBI-Genbank sequence entryS74166:

(P033) SEQ ID NO. 37 5′-gcaCTCGAGgggtagatgcagcctgtgttccgtttactg-3′, and(P034) SEQ ID NO. 38 5′-gctCTCGAGCATGCctgagcccaccaggaagtcctctgtg-3′

Restriction enzyme sites appended to the 5′ termini of each primer arehighlighted in uppercase letters. In addition to XhoI restriction sites(CTCGAG) present in both primers, the reverse primer P034 contains anadditional SphI restriction site (GCATGC, underlined) containing anadditional unique restriction site required for later cloning purposes.PCR amplification with primer pair P033 and P034 on genomic mouse DNAwill result in a 466 by PCR fragment that can be digested with therestriction enzyme XhoI and which is then ligated into XhoI linearizedvector pAB-3. A clone with the HS4 insert in the desired orientation isdetermined by diagnostic restriction enzyme digestion and DNAsequencing, and is designated as vector construct pAB-4 (FIG. 12 b). Thelast 3′α enhancer fragment, HS3, can be PCR amplified using mousegenomic DNA as a template with the following primers designed accordingto published NCBI-Genbank sequence entry X96607 (recognition sequencesfor the restriction enzyme SphI are again highlighted in uppercaseletters):

(P035) SEQ ID NO. 39 5′-ttaaGCATGCaaccacatgcgatctaagggatattgggg-3′ and(P036) SEQ ID NO. 40 5′-ttaaGCATGCgatcattgagctccggctctaacaactggg-3′

The PCR amplified HS3 3′α enhancer region is digested with SphIrestriction enzyme and ligated into SphI linearized vector pAB-4 inorder to generate pAB-5. Any of the vectors pAB-3, 4, or 5 can be usedfor inserting the coding regions for heterologous immunoglobulin heavychains (FIG. 12 b).

Each of the human immunoglobulin isotypes can be expressed as a membranebound surface immunoglobulin or as a secreted antibody, which isregulated by alternative splicing events either maintaining or deletingthe membrane spanning exons from the mRNA transcript. Although eachimmunoglobulin isotype has its own coding region for the membranespanning exons, the membrane regions of all human IgG, IgA and IgEantibodies are virtually identical for the individual subtypes.Therefore, the same IgG membrane exons can be employed for membraneanchoring IgG1, IgG2, IgG3 and IgG4 antibodies, a different set of exonsare used for both IgA1 and IgA2, and each different exons are requiredfor each IgE and IgM antibodies.

Therefore, the next step in the cloning procedure towards the generationof recombinant retroviral Ig expression constructs is the insertion ofthe various membrane spanning regions for human IgG, IgA, IgM and IgEantibodies into any of the vectors pAB-3, 4, or 5. As a representativeexample, the further cloning procedures are described for the basicretroviral construct pAB-3 containing the V_(H)promoter, the EμHenhancer and the minimal 3′αE enhancer (HS1,2) elements (FIG. 12 c).

The μH chain membrane spanning region is encoded by two exons, μM1 andμM2, located 1.85 kb downstream of the C_(μ)H constant region exonsC_(μ)H₁-C_(μ)H₄. The μM1 and μM2 exons including the endogenouspolyadenylation site are contained within a genomic fragment of 773 bpthat can be amplified from mouse genomic DNA by PCR using the followingprimers designed based on published NCBI-Genbank contig sequenceNT_(—)010168:

(P037) SEQ ID NO. 41 5′-catACGCGTcgctgcatcaggctttcaggggcccagccc-3′ and(P038) SEQ ID NO. 42 5′-catACGCGTccctcaccagaaagcagtttcatggataaaatg-3′

The MluI restriction sites appended to the 5′ end of each primer arehighlighted in uppercase letters. The resulting PCR product can bedigested with MluI restriction enzyme and the resulting 773 bp fragmentis ligated into MluI linearized vector pAB-3. A clone containing the μM1and μM2 exons in correct orientation and DNA sequence as verifiedrestriction enzyme mapping and by DNA sequencing is designated constructpAB-3.1 (FIG. 12 c).

Membrane deposition of IgG antibodies can e.g. be accomplished by themembrane spanning exons of the IgG₁ gene. The IgG₁ membrane spanningregion is encoded by two exons located 1.25 kb downstream of the C_(γ1)Hexons spanning a region of 1.9 kb. In order to shorten the intronbetween γ1M1 and γ1M2, a single overlap extension (SOE) PCR approach isused, by which the two exons are fused to a smaller PCR fragment.Generally, the fusion of two PCR products can be achieved, if thereverse primer of the upstream PCR product and the sense primer of thedownstream PCR product exhibit roughly 30-40 by of complementarity.These regions of perfect complementarity can be appended as 15-20nucleotides to the 5′ ends of the reverse primer and forward primersused for the amplification of the upstream and the downstream PCRproducts, respectively. However, in case of the fusion of the two γ1M1and γ1M2 exons, by coincidence, two completely identical sequences of 28bp are present 78 bp downstream of γ1M1 and 256 bp upstream of γ1M2exon. These sequences can be used to generate a shorter PCR fragment bySOE-PCR containing both the γ1M1 and γ1M2 exons for the membranespanning region of γH chains. Therefore, two PCRs are performed inparallel on human genomic DNA using the following primers designed basedon the published NCBI-Genbank sequence AL122127. A PCR fragment of 315basepairs containing the γ1 membrane spanning exon 1 (γ1M1) is amplifiedfrom human genomic DNA using primers:

(P039) SEQ ID NO. 43 5′-catACGCGTacagagggaatcacccccagaggcccaagccc-3′ and(P040) SEQ ID NO. 44 5′-gacagcgtcagggacaggtggggacagc-3′,with primer P039 binding to position 9710-9741, and primer P040 bindingto position 9435-9462 of NCBI-Genbank sequence entry AL122127.

A 555 basepair PCR fragment containing the membrane spanning exon 2(γ1M2) can be amplified from human genomic DNA using primers:

(P041) SEQ ID NO. 45 5′-gctgtccccacctgtccctgacgctgtc-3′, and (P042) SEQID NO. 46 5′-catACGCGTttatttggaaggggggcgtgtcaggtgtgtcagggtc- 3′with primer P041 binding to position 8137-8164, and primer P042 bindingto position 7624-7654 of NCBI-Genbank sequence entry AL122127. The twoPCR products can then be fused to a 822 by PCR fusion product by mixing1/100 of the γ1M1 exon PCR product with 1/100 of the γ1M2 exon PCRproduct and repeating a PCR with sense and reverse primers P039 andP042. In addition to MluI restriction enzyme recognition sites presentin both primers P039 and P042 (highlighted in uppercase letters), whichare required for the cloning of the SOE fusion PCR product, primer P042additionally contains the complementary sequence of an artificialpolyadenylation signal (underlined) allowing correct transcriptionaltermination of the immunoglobulin transcript in the final retroviralimmunoglobulin expression vector. The 832 bp γ1M1γ1M2-polyA signal PCRfragment is then digested with MluI restriction enzyme and is ligatedinto vector pAB-3. A clone with the correct orientation of the insertverified by restriction enzyme mapping and DNA sequencing is designatedclone pAB-3.2 (FIG. 12 c).

Cloning of the al membrane spanning coding region can be achieved by PCRamplifying from human genomic DNA a fragment containing the single exonfor α1M including its endogenous polyA site. The following primers canused for this PCR amplification, designed according to the publishedNCBI-Genbank sequence entry M60193:

(P043) SEQ ID NO. 47 5′-atcACGCGTctcaggccttagatggggacccagaccc-3′ and(P044) SEQ ID NO. 48 5′-attACGCGTgacccccgtctcctcattcagagtctgtg-3′

Both primers contained MluI cloning sites appended to the 5′ end of eachprimer (highlighted in uppercase letters) and will amplify a PCR productof 768 basepairs that can be digested with MluI restriction enzyme thatis then ligated into MluI linearized pAB-3. A clone with correctorientation of the insert verified by restriction enzyme mapping and DNAsequencing is designated clone pAB-3.3.

For the expression of membrane bound IgE antibodies the membranespanning region for ε₁H chains, which is encoded by two exons, ε₁M1 andε₁M2 is PCR cloned. For this a 858 by PCR product is amplified fromhuman genomic DNA using the following primers designed according topublished NCBI-Genbank sequence entry X63693:

(P045) SEQ ID NO. 49 5′-catACGCGtcgggacctgggtgcccaccctcagggctgg-3′ and(P046) SEQ ID NO. 50 5′-aatACGCGTttatttgtgccctgggctgggtgccgggccctccttgg-3′

Both primers contain MluI cloning sites appended to the 5′ end of eachprimer (highlighted in uppercase letters) resulting in a 858 by PCRproduct that can be digested with MluI restriction enzyme. In addition,the reverse primer P046 is designed to contain the complementarysequence of an artificial polyA signal (underlined) ensuring propertermination of mRNA transcription in the final expression construct. TheMluI digested PCR fragment is ligated into MluI linearized pAB-3. Aclone with correct orientation of the insert verified by restrictionenzyme mapping and DNA sequencing is designated clone pAB-3.4.

Following cloning of the various membrane spanning exons for IgG, IgA,IgM and IgE isotypes, the exons for the various human constant regionshave to be inserted into retroviral constructs pAB-3.1 to 3.4. For this,DNA fragments encoding the constant region exons are cloned into theunique NotI restriction enzyme recognition site of each of the pAB-3.1to 4 constructs.

Cloning of retroviral expression vectors for all immunoglobulin H chainisotypes, γ1, γ2, γ3 and γ4 is depicted in FIG. 12 d. The followingprimers can be used for the PCR amplification of the four different IgGheavy chain subtypes using human genomic DNA as a PCR template:

For γ1 (primers designed according to published NCBI-Genbank sequenceAL122127):

(P047) SEQ ID NO. 51 5′-gcatGCGGCCGCcctgggcccagctctgtcccacacc-3′, and(P048) SEQ ID NO. 52 5′-gcatGCGGCCGCtgggtgctttatttccatgctgggtg-3′

For γ2 (primers designed according to published NCBI-Genbank sequenceJ00230):

(P049) SEQ ID NO. 53 5′-tataGCGGCCGCagggagtgggctaaggtgaggcaggtgg-3′ and(P050) SEQ ID NO. 54 5′-attaGCGGCCGCctcagactcggcctgacccacggaaagaac-3′

For γ3 (primers designed according to published NCBI-Genbank sequenceAL122127):

(P051) SEQ ID NO. 55 5′-gcatGCGGCCGCccagctctgtcccacaccgcagtcacatgg-3′and (P052) SEQ ID NO. 565′-gctaGCGGCCGCtcgcaggggcccagggcagcgctgggtgctt-3′

For γ4 (primers designed according to published NCBI-Genbank sequenceK01316):

(P053) SEQ ID NO. 57 5′-attaGCGGCCGCggatagacaagaaccgaggggcctctg-3′ and(P054) SEQ ID NO. 58 5′-taatGCGGCCGCccagggcagtggtgggtgctttatttcc-3′

In each case, the PCR products of 1788 bp for γ₁H of 1959 bp for γ₂H, of2374 bp for γ₃H, and of 1820 bp for γ₄H can be digested with NotIrestriction enzyme, upon which the digested fragment is ligated intoNotI linearized pAB-3.2 (FIG. 12 d). Constructs containing the insertsin correct orientation and without any mutations as determined byrestriction enzyme mapping and DNA sequencing are designated pAB-3.2-G1,pAB-3,2-G2, pAB-3.2-G3, and pAB-3.2-G4, respectively.

For the cloning of the constant region exons encoding μH, α1H, α2H, andεH chains, identical method are employed (FIG. 12 e). The followingprimers are used for the PCR amplification of the different IgH isotypesusing human genomic DNA as a template:

For μH (primers designed according to published NCBI-Genbank sequenceAB019441):

(P055) SEQ ID NO. 59 5′-attaGCGGCCGCcaccccagtaggccagagcatcgtgcac-3′ and(P056) SEQ ID NO. 60 5′-taatGCGGCCGCcacccctgatagccatgacagtctggg-3′

For α₁H and α₂H (primers designed according to published NCBI-Genbanksequences J00220, and J00221):

(P057) SEQ ID NO. 61 5′-attaGCGGCCGCtgtcaccaggcctctctgtgctgggttcc-3′ and(P058) SEQ ID NO. 62 5′-tattGCGGCCGCcggatggaagcgtggggctgcttgggg-3′

For εH (primers designed according to published NCBI-Genbank sequenceL00022):

(P059) SEQ ID NO. 63 5′-taatGCGGCCGCcacggggtccccagctcccccatccagg-3′ and(P060) SEQ ID NO. 64 5′-atatGCGGCCGCtcccaagaatgggtgtactggggctc-3′

In each case, the PCR products of 2229 bp for μH, of 1667 bp for α₁H andα₂H, and of 1908 bp for εH can be digested with NotI restriction enzymeand can be ligated into NotI linearized pAB-3.1, pAB-3.3, and pAB-3.4,respectively (FIG. 12 e). Constructs containing the inserts in correctorientation and without any mutations as determined by restrictionenzyme mapping and DNA sequencing are designated pAB-3.1-M, pAB-3.3-A1,pAB-3.3-A2, and pAB-3.4-E, respectively.

The different retroviral H chain expression constructs controlled bynatural IgH chain promoter and enhancer elements still require theinsertion of a variable domain coding region, which will be presentedafter the description of the methods for cloning of the retroviral IgLchain expression vectors, because similar strategies for expressingunique or diverse specificities will be presented for both the completeretroviral IgH and IgL chain expression constructs.

(b) Generation of Retroviral Constructs for the Regulated Expression ofIgκL Chains

Similarly to the constructs designed for IgH chain expression,retroviral constructs designed for the differentiation stage specificexpression of IgκL chains require the presence of defined κL chain locusspecific promoter and enhancer elements. These include a Vκpromoter, theκ light chain intron enhancer (κiE), and the κ3′ enhancer (κ3′E)elements. The sequential cloning of these elements can be accomplishedas follows (FIG. 13 a,b).

A mouse Vκpromoter is PCR amplified as a 290 bp PCR fragment from mousegenomic DNA using the following primers (designed according to thepublished NCBI-Genbank sequence entry X52768):

(P061) SEQ ID NO. 65 5′-ccATCGATcctggtctacagtgtgaggtactggac-3′, and(P062) SEQ ID NO. 66 5′-ccATCGATGGATCCtctgagagctggaagagaggatgctttattagg-3′

Restriction sites appended to the 5′ end each primer are highlighted byuppercase letters. In addition to ClaI sites present at the 5′ ends ofboth primers, the reverse primer P062 contains an additional BamHIrestriction site (GGATCC, underlined) introducing another uniquerestriction site into the PCR product which will be used for latercloning purposes. After ClaI restriction enzyme digestion of the PCRfragment, the amplified V_(κ) promoter is ligated into ClaI linearizedpLIB vector. Ligation products containing the V_(κ) promoter in thedesired orientation (opposite to the transcriptional orientation of the5′LTR promoter) are identified by diagnostic restriction enzyme mappingand DNA sequencing giving rise to construct pAB-6 (FIG. 13 a).

The κiE and the coding region for the κL chain constant domain arelocated in close proximity in both the human and the mouse κL chain genelocus and the enhancer appear to function across species. Therefore, theoptional cloning procedures for two different retroviral expressionvectors encoding κL chains is described, one utilizing the human, andthe other utilizing the murine κ intron enhancer (κiE). It should benoted, that both constructs can be used interchangeably. Cloning of theconstruct containing the human κiE can be achieved by amplifying a PCRfragment from genomic human DNA containing both the human κiE and thehuman Cκ exon including a 5′ located MAR using the following primers(designed based on published NCBI-Genbank sequence entry AF017732):

(P063) SEQ ID NO. 67 5′-cgGGATCCTGACGCGTaccagggagaagactgatttattagagatttc-3′, and (P064) SEQ ID NO. 685′-gatGCGGCCGCaaagattcactttatttattcattctcc-3′

Additional restriction sites appended to the 5′ end of each primer areagain highlighted in uppercase letters (BamHI in primer P063 and NotI inP064). Primer P063 contains the sequence for an additional MluIrestriction site used in later cloning steps. The PCR primers amplify a2359 by fragment that can be digested with BamHI and NotI, and which canthen be directionally cloned into BamHI-NotI linerized pAB-6 generatingretroviral construct pAB-7 (FIG. 13 a).

For the construct utilizing the murine κiE a PCR product containing themurine κiE is fused to a PCR product containing the human Cκ region bythe SOE-PCR approach described previously. For this, first PCRamplification of the murine κiE and the human Cκ region is performed inparallel, with the κiE reverse primer and the Cκ region forward primerexhibiting a 36 bp region of complementarity. In the second PCR, whichis used to fuse these two PCR products, 1/100 of each PCR reaction ismixed and amplified with the κiE forward (P065) and the Cκ regionreverse primer (P064) resulting in the fusion of the two PCR products atthe region of 36 bp terminal complementarity.

The following primers can be used for the amplification of the murineκiE (designed based on the published NCBI-Genbank sequence entry V00777)using mouse genomic DNA as a template:

(P065) SEQ ID NO. 69 5′-cgGGATCCAAGCTTgaaaaatgtttaactcagctactataatccc-3′ (P066) SEQ ID NO. 70 5′-gttttgttggagctcccctttgaagatattctcaggcttccttc-3′

The following primers can be used for the amplification of the human Cκregion (designed based on the published NCBI Genbank sequence entryAF017732) using human genomic DNA as a template:

(P067) SEQ ID NO. 71 5′-aatatcttcaaaggggagctccaacaaaacaatttagaactttattaag-3′ (P064) SEQ ID NO. 685′-gatGCGGCCGCaaagattcactttatttattcattctcc-3′

The regions of terminal complementarity in primers P066 and P067 areunderlined, which mediate the fusion of the two PCR products in a secondPCR carried out with primers P065 and P064 and a mixture of the twofirst PCR products as a template. The resulting murine κiE-human Cκfusion PCR product of 1524 bp can be digested with BamHI and NotI and isthen directionally cloned into BamHI-NotI linearized vector pAB-6generating vector pAB-8 (FIG. 13 a).

Next the murine κ3′E is cloned downstream of the human Cκ regions inboth pAB-7 and pAB-8 generating constructs pAB-7.1 and pAB-8.1,respectively. The primers used for the PCR amplification for the κ3′Efrom mouse genomic DNA are as follows (designed based on publishedNCBI-Genbank sequence entry X15878):

(P068) SEQ ID NO. 72 5′-acgcGTCGACtagaacgtgtctgggccccatgaaacatc-3′, and(P069) SEQ ID NO. 73 5′-cgatGTCGACagctcaaaccagcttaggctacacagagaaac-3′

Both primers contain SalI restriction sites (highlighted in uppercaseletters), such that the resulting 827 bp PCR product can be cloned intothe compatible SalI restriction sites of pAB-7 and pAB-8. Vector clonescarrying the insert in the correct orientation and with the correct DNAsequence, as determined by restriction enzyme mapping and DNAsequencing, are designated pAB-7.1 and pAB-8.1 (FIG. 13 a).

In a next cloning step, a puromycin resistance marker under control of aconstitutive SV40 promoter is cloned into the retroviral κL chainexpression constructs. The inclusion of an expression cassette forpuromycin resistance is preferred, because both IgH and IgL chainretroviral constructs need to be stably co-transduced into murine preBcells in order to allow expression of complete heterologous antibodies.In order to increase the efficiency of retroviral cotransduction,retroviral IgL chain constructs are transduced first, and stablytransduced cells are selected for the integration of the IgL chainconstructs using puromycin selection. A secondary transduction of IgHchain retroviral vectors will then give rise to cells with the potentialfor antibody production in each cell that is transduced with an IgHchain retroviral vector.

The puromycin expression cassette can be cloned from the puromycinresistance vector pPur (Clontech) by SOE-PCR deleting some unwantedinternal restriction enzyme sites and adding suitable ClaI cloning sitesto the ends of the amplified PCR fragment.

The primers used for the SOE fusion PCR are:

(P070) SEQ ID NO. 74 5′-ccATCGATctgtggaatgtgtgtcagttagggtgtgg-3′ (P071)SEQ ID NO. 75 5′-tcaggggatggtggcggccctaggcctccaaaaaagcctcctca ctacttc-3′(P072) SEQ ID NO. 76 5′-cttttttggaggcctagggccgccaccatcccctgacccacgcccctgacc-3′ (P073) SEQ ID NO. 77 5′-ccATCGATccagacatgataagatacattgatgag-3′

First, two PCR amplifications are performed in parallel with primerpairs P070/P071 and P072/P073 using the same pPur template for the PCRs.Reverse primer P071 and forward primer P072 are designed to contain aregion of 36 bp perfect complementarity (underlined), which will allowthe fusion of the two PCR products by a second PCR using aliquots of theprevious PCR fragments in an additional PCR amplification using primersP070 and P073 alone. The latter primers contain ClaI restriction sites(uppercase letters) allowing the cloning of the resulting 1351 bppuromycin expression cassette into compatible ClaI sites of pAB-7.1 andpAB-8.1. This generates retroviral constructs pAB-7.1Pur and pAB-8.1Pur,respectively (FIG. 13 b), which are the constructs into which singleVJ_(κ) regions or VJ_(κ) region libraries encoding unique or diversevariable domains of κL chains, respectively, can be inserted. Theprocedure for cloning of the variable region exons will be presentedfurther below together with the cloning of H chain and λL chain variableregions, after also the cloning of the retroviral expression vectors forheterologous, human λL chains have been described.

(c) Generation of Retroviral Constructs for the Regulated Expression ofIgλL Chains

In some cases it is desirable to produce heterologous antibodiescontaining λL chains instead of κL chains. Although it might besufficient to replace the human Cκ coding region in the retroviralconstructs pAB-7.1Pur and pAB-8.1Pur with any of the human Cλ codingregions, a perfect endogenous λL chain expression pattern will only beachieved by controlling the expression of λL chains by λL chain genelocus specific promoter and enhancer elements. This is particularly truein the murine system, where κL chain gene rearrangement and expressionprecedes the rearrangement and expression of λL chains during B celldifferentiation.

The murine λL chain gene locus specific enhancer elements are the λ2-4enhancer and the λ3-1 enhancer, which are located close to the Cλ2, Cλ4,and Cλ1, Cλ3 constant region exons and regulate the expression of λ2,λ4, and λ1, λ3 light chain isotypes, respectively.

As in the case of κL chain retroviral expression constructs (see above),retroviral λL chain expression vectors are designed to contain apuromycin resistance gene expression cassette. Therefore, in the firstcloning step, a SV40 promoter controlled puromycin expression cassetteis fused to a Vλ promoter by SOE-PCR using mouse genomic DNA andpAB-7.1Pur as templates for PCR amplification, respectively. The primersfor the two parallel SOE-PCR reactions are as follows (with the Vλpromoter specific primers P076 and P077 designed based on the publishedNCBI-Genbank database sequence entry J00591):

(P074) SEQ ID NO. 78 5′-ccATCGATctgtggaatgtgtgtcagttagggtgtgg-3′ (P075)SEQ ID NO. 79 5′-gatgtcacgtgagatcccagacatgataagatacattgatga gtttg-3′(P076) SEQ ID NO. 80 5′-tcttatcatgtctgggatctcacgtgacatcttataataaacctg-3′ (P077) SEQ ID NO. 81 5′-ccATCGATACGCGTaattcacaaaccaagtctattattttcaata-3′

As for the SOE fusion PCRs described earlier, first two PCRamplifications are performed in parallel, i.e. the puromycin expressioncassette with primers P074 and P075 on pAB-7.1Pur as a template, and theVλ promoter with primers P076 and P077 on mouse genomic DNA as atemplate. Each 1/100 volume aliquots of the first parallel PCRs are thenmixed and amplified again with primers P074 and P077. The puromycinexpression cassette is thereby fused to the 5′ end of the Vλ promoter,because of the 36 bp designed perfect complementarity in reverse primerP075 and sense primer P076 (underlined) that is used for the first roundparallel PCRs.

Both primers P074 and P077 contain ClaI restriction sites that can beused to clone the PCR fusion product into ClaI linearized pLIB. Anadditional MluI restriction site (underlined) is also incorporated intoreverse primer P077 giving rise to an additional unique MluI site 3′ ofthe Vλ promoter required for further cloning steps. A vector clone withthe puromycin-Vλ promoter fusion PCR fragment inserted in pLIB in thecorrect orientation and with the correct DNA sequence, as verified byrestriction enzyme mapping and DNA sequencing, is designated vectorpAB-9 (FIG. 14 a).

Next, the two murine A2-4E and λ3-1E enhancer elements are PCR amplifiedfrom mouse genomic DNA, again followed by fusing both sequences bySOE-PCR. The following primers can be used (based on the publishedNCBI-Genbank sequences entries X54550 and X54608, respectively):

(P078) SEQ ID NO. 82 5′-catGCGGCCGCgagtatccctgtgcagtggggatactcag-3′(P079) SEQ ID NO. 83 5′-tctcaggagagaCTCGAGactcctttgtgctctgatagcacacatgac-3′ (P080) SEQ ID NO. 845′-gcacaaaggagtCTCGAGtctctcctgagatggttcataggcc tgcc-3′ (P081) SEQ ID NO.85 5′-ggGAATTCtctagacataaggaacaaagtcagtgtgcc-3′

Like in the previous approach, first, two PCR fragments are amplified inparallel with primer pair P078/P079 and P080/P081 using mouse genomicDNA as a template, and in a second PCR round aliquots of these PCRproducts are used as a template and fused by PCR amplification withprimers P078 and P081 due to the 36 bp complementarity of primers P079and P080 (underlined).

The resulting 1513 bp PCR fusion product can be digested with NotI andEcoRI restriction enzymes (the respective recognition sites beingincorporated into primers P078 and P081; highlighted in uppercaseletters) and is directionally cloned into NotI/EcoRI linearized pAB-9. Avector clone with the correct DNA sequence, as verified by restrictionenzyme mapping and DNA sequencing, is designated vector pAB-10 (FIG. 14a). The complementary regions in primers P079 and P080 are designed tocontain an additional unique XhoI site (highlighted in uppercaseletters), in order to allow the separate removal of each of the enhancerelements, if desired.

In a next step, any of the human Cλ regions is cloned into theretroviral construct pAB-10 using the following primers (designed basedon published NCBI-Genbank sequences D87023 and D87017):

(P082) SEQ ID NO. 86 5′-ccACGCGTaaaagcttgtctaggtggagcccactccttgcc-3′(P083) SEQ ID NO. 87 5′-catGCGGCCGccctgtaccccacactgagaaccccgggg-3′

These primers are able to amplify any of the functional human Cλ regionsas 1094 bp PCR fragments if human genomic DNA is used as a template.Primers P082 and P083 contain restriction sites for ClaI and NotI,respectively, allowing ClaI-NotI double digestion of the PCR fragmentsand their directional ligation into pAB-10 in order to generate(depending on the amplified Cλ region) either pAB-11-λ1, pAB-11-λ2,pAB-11-λ3, or pAB-11-λ7 (FIG. 14 b).

For the generation of a complete IgλL chain retroviral expression vectora VλJλ rearranged variable region exon has to be cloned into the uniqueMluI and HindIII sites of the various pAB-11 constructs, which isdescribed in the following section, together with the generation ofcomplete IgH and IgκL chain retroviral expression vectors.

Example 7 Cloning of V_(H)DJ_(H) or V_(L)J_(L) Variable Region Exonsinto Retroviral IgH and L Chain Expression Constructs

The retroviral expression constructs for the different human IgH and IgLchains described so far contain all the coding regions and controlelements for human antibody expression, with the exception of the codingregions for the variable domains. Diverse repertoires. (libraries) ofexons encoding variable region domains can e.g. be cloned from genomicDNA of human peripheral blood lymphocytes containing B lymphocytes withdiverse V_(H)DJ_(H) and V_(L)J_(L) DNA rearrangements. These codingregions for the variable domains need to contain a common leader exon,located 5′ of each V_(H)DJ_(H) and V_(L)J_(L) rearranged exon (FIG. 15),which is required for the proper transport of IgH and IgL chains throughthe endoplasmatic reticulum, the trans golgi network and eventually tothe cell surface. Degenerate primers binding to the majority of leadersequences of the human IgH, IgκL and IgλL variable region leadersequences have been described and are used in combination withdegenerate primers amplifying the various human J_(H) and J_(L) genesegments for the cloning of the V_(H)DJ_(H) or V_(L)J_(L) variablecoding regions. Recognition sites for restriction enzymes used for thedirectional cloning of the PCR products (see FIG. 15) are appended tothe 5′ end of the degenerate primers and are highlighted in uppercaseletters. Positions in the degenerate primers, which may contain morethan only one nucleotide are indicated in parentheses.

Forward primers for the PCR amplification of human Ig heavy chainvariable domains including leader sequences from human genomic DNA ofperipheral B lymphocytes are as follows:

(P084) SEQ ID NO. 88 5′-gcGGATCCatggactggacctggagg(ag)tc(ct)tct(gt)c-3′(P085) SEQ ID NO. 89 5′-gcGGATCCatggag(ct)ttgggctga(cg)ctgg(cg)ttc(ct)t-3′ (P086) SEQ ID NO. 905′-gcGGATCCatgga(ac)(ac)(at)act(gt)tg(gt)(at)(cgt)c(at)(ct)(cg)ct(ct)ctg-3′

A forward primer for the amplification of human Igκ light chain variabledomains including leader sequence from human genomic DNA of peripheral Blymphocytes is as follows:

(P087) SEQ ID NO 91 5′-gcGGATCCatggacatg(ag)(ag)(ag)(agt)(ct)cc(act)(acg)g(ct)(gt)ca(cg)ctt-3′

A forward primer for the amplification of human Igλ light chain variabledomains including leader sequence from human genomic DNA of peripheral Blymphocytes is as follows:

(P088) SEQ ID NO 92 5′-gcACGCGTatg(ag)cctg(cg)(at)c(ct)cctctc(ct)t(ct)ct(cg)(at)(ct)-3′

Note, that instead of a BamHI restriction enzyme recognition siteappended to the 5′ end of primers P084-P087 for cloning purposes, arecognition site for the BglII restriction enzyme can be used instead(compare to FIG. 15), because BglII generates the same compatibleoverhangs, as BamHI. In case of cloning of VλJλ regions the forwardprimer P088 contains a MluI site, because the retroviral constructaccepting the fragment already contains internal BamHI restrictionsites. Alternatively, a primer similarly to P088 may be used containingan AscI site producing the same CGCG overhang as MluI restrictionenzyme.

Reverse primers for the amplification of all six human IgH J chain genesegments: are as follows. HindIII sites, highlighted in uppercaseletters, are appended to the 5′ ends of the primers including someflanking nucleotides for cloning purposes.

A reverse primer for the PCR amplification of variable region exonscontaining J_(H)1, J_(H)4, and J_(H)5 gene segments using genomic DNAfrom human B lymphocytes is as follows:

(P089) SEQ ID NO 93 5′-tatAAGCTTacctgaggagacggtgaccagggtgccctgg-3′

A degenerate reverse primer for the amplification of variable regionexons containing J_(H)2, J_(H)3, and J_(H)6 gene segments using genomicDNA from human B lymphocytes is as follows:

(P090) SEQ ID NO 94 5′-tatAAGCTTacctgaggagacggtgacca(tg)(tg)gt(gc)cc(ta)(tc)(gt)g-3′

A reverse degenerate primer for the amplification of variable κ regionexons containing human Jκ1-4 gene segments using genomic DNA from humanB lymphocytes is as follows:

(P091) SEQ ID NO 95 5′-tatAAGCTTacgtttgatctcca(cg)(ct)ttggtccct(tgc)ggcc-3′

A reverse primer for the amplification of variable κ region exonscontaining the human Jκ5 gene segment using genomic DNA from human Blymphocytes is as follows:

(P092) SEQ ID NO 96 5′-tatAAGCTTacgtttaatctccagtcgtgtcccttggcc-3′

A reverse degenerate primer for the PCR amplification of variable regionIgλL exons containing Jλ1-3 gene segments using genomic DNA from human Blymphocytes is as follows:

(P093) SEQ ID NO 97 5′-tatAAGCTTacctaggacggtcagcttggtccc(ta)(cg)c(tg)cc-3′

A reverse primer for the PCR amplification of variable region IgλL exonscontaining Jλ7 using genomic DNA from human B lymphocytes is as follows:

(P094) SEQ ID NO 98 5′-tatAAGCTTaccgagggcggtcagctgggtgcctcctcc-3′

For the PCR amplification and cloning of variable domain exons containrearranged V_(H)DJ_(H) and V_(L)J_(L) gene segments with the abovedescribed primers P084-P094, equimolar amounts of primers are used, ifmore than one forward or reverse primer is indicated for a particularvariable region. In case of cloning of variable regions for H and κLchains, the PCR fragments are digested with BamHI and HindIII and aredirectionally cloned into BamHI/HindIII double digested vectors for IgHand IgκL chain expression. In case of cloning of variable regions for λLchains, the PCR fragments are digested with MluI and HindIII and aredirectionally cloned into MluI/HindIII double digested vectors for IgλLchain expression.

Example 8 Sequential Transduction of Retroviral Expression Vectors forHeterologous IgH and L Chains into Genetically Modified Murine preBCells

The retroviral transduction of stromal cell and IL7 dependent murinepreB cells has been described in detail in Example 3. For the sequentialtransduction of retroviral expression constructs encoding IgH and IgLchains, identical protocols are used for the transfection of theecotropic packaging cells and the conditions to infect recombinantretroviruses into preB cells. The only difference is that two retroviralexpression constructs are sequentially transduced by the protocoloutlined below.

Stromal cell and IL7 dependent preB cells are first infected withsupernatant from GPE packaging cells that have transiently beentransfected with the retroviral construct encoding heterologous IgLchains. As described above, the IgL chain expression vectorsadditionally contain a puromycin resistance marker. Therefore, stablytransduced cells can be selected by the addition of 2 μg/ml puromycin tothe tissue culture medium 24 hours post transduction. Transduced cellsare cultured and expanded for an additional period of 48 hours inpuromycin containing medium. Non-transduced preB cells will beeliminated due to the puromycin selection and a 100% IgL chaintransduced population of preB cells will be obtained after 48 hours ofculture.

At this point, cells are incubated with supernatant of the ecotropicpackaging cell line GPE that have transiently been transfected withretroviral vectors encoding heterologous IgH chains. These cells arethen kept in culture for an additional period of 24 hours upon whichthey cease to proliferate due to the expression of the heterologousimmunoglobulin heavy chains. The cells are then harvested and may beused for the transplantation of immunocompromised mice, where thesecells can reconstitute B lineage compartments, and where they are ableto participate within an immune response giving rise to heterologousantibody secreting cells.

Example 9 Transplantation of Long-Term Proliferating Murine Precursor BCells into Mice for the Production of Heterologous Antibodies

Stromal cell and IL7 dependent preB cells expressing heterologous IgHand L chains from stably transduced retroviral expression vectors areintravenously injected into either B cell deficient JHT mice, ortogether with CD4⁺ T-helper cells into CB17 SCID, RAG-2 or RAG-2deficient mice, such that the transplanted cells can mount T celldependent or independent immune responses upon antigenic stimulation ofthe transplanted mice.

For this, 1.5 to 3 month old mice are sublethally irradiated with300-600 rad γ-irradiation and 4-6 hour post irradiation the mice areintravenously injected with 5×10⁶ to 10⁷ preB cells resuspended in PBS.In case of additional transplantation of CD4⁺ T-helper cells, CD4⁺ cellsare isolated from pooled cervical, axillary, mesenteric and inguinallymph nodes of syngeneic mice by depletion of B cells and CD8⁺ T cellsby use of sheep-anti-mouse Ig antibody coated Dynabeads (Milan AnalyticaAG, La Roche, Switzerland). 10⁶ of these routinely more than 90% pureCD4⁺T-helper cells are coinjected with preB cells. Cell populations areallowed to reconstitute B and T cell compartments of the transplantedhosts for 6 weeks following transplantation.

Example 10 Immunization of preB Cell Grafted Mice

Immunizations of immunocompromised mice transplanted and reconstitutedwith HupreB cells and T helper cells is done essentially as described(Harlow and Lane, Cold Spring Harbor Laboratory Press, p. 150-173,1988). In brief, 10-50 μg of antigen is dissolved in 250 μl PBS andvigorously mixed with 250 μl complete Freund's adjuvant and theantigen-adjuvant mixture is injected intraperitonally. After two weeks,mice are boosted intraperitonally with 5-20 μg of antigen, this timemixed with incomplete Freund's adjuvant. 10 days following the firstboost, mice are analyzed for antigen specific heterologous antibodies inthe serum. The best responders are boosted again four weeks afterwardswith 5-20 μg of antigen in incomplete Freund's adjuvant bothintraperitoneally and intravenously. 3 days later, spleen cells areisolated from mice and used for the generation of hybridoma cellssecreting heterologous antigen-specific monoclonal antibodies.

Example 11 In Vitro Stimulation of HupreB Cells with for theDifferentiation into Antibody Secreting Plasma Cells

Stromal cell and IL7 dependent HupreB cells can also be differentiatedinto antibody secreting cells entirely in tissue culture in vitro. Forthis, continuously proliferating HupreB cell cultures are firstdifferentiated into surface immunoglobulin positive B cell by withdrawalof IL7 from the tissue culture medium and the continued culture onirradiated stromal cells for two to three days. The absence of growthfactors in the medium results in the arrest of proliferation and theinduction of differentiation which is accompanied by the induction ofapoptosis, unless an anti-apoptotic gene, like bcl-2 is expressed in thedifferentiating cells.

The differentiated cells are then harvested, and replated on freshirradiated stromal cells e.g. in the presence of 100 U/ml rIL4 and 20μg/ml of an agonistic anti-CD40 monoclonal antibody. Within 5-6 days ofculture the cells differentiate into large proliferating cells of plasmacell phenotype, that are able to secrete large amounts of antibodiesfrom the heterologous Ig gene loci and that can be used for generationof heterologous monoclonal antibody secreting hybridoma cell clones.

Example 12 Fusion of Myeloma Cells with Plasma Cells Derived from HupreBCells for the Generation of Hybridomas Producing Human MonoclonalAntibodies

For the immortalization of plasma cells derived from HupreB cells,either upon in vitro differentiation (Example 11), or after immunizationof transplanted mice (Example 10), the plasma cells need to be fusedwith an immortal myeloma cell line. The myeloma cell lines that are usedfor the cell fusion lack the expression of the HGPRT gene, involved inthe salvage pathway of nucleotide biosynthesis. While these cells growindefinitively in regular tissue culture medium, they will die, if denovo nucleotide synthesis is blocked by addition of e.g. the drugaminopterine to the tissue culture medium. In contrast, cells expressingthe HGPRT gene can survive a block of de novo nucleotide synthesis byaminopterine, especially, if additional thymidine and hypoxanthine aresupplemented, because the HGPRT gene product can utilize thesesubstances for the synthesis of pyrimidine and purine nucleotides,respectively. Thus, in HAT selection medium (containing hypoxanthine,aminopterine and thymidine), only those cells survive and proliferate,that resulted from a fusion of the mortal, but HGPRT⁺, antibodysecreting plasma cell and the immortal, but HGPRT, and thereforeaminopterine sensitive myeloma cell. These cells are called hybridomacells, and, if subcloned, will secrete a monoclonal antibody of thespecificity encoded by the plasma cell fusion partner.

For the generation of hybridoma cells, either plasma cells from thespleen of immunized mice transplanted with HupreB cells, or plasma cellsgenerated in vitro by anti-CD40 and IL4 stimulation are used. In eachcase a homogeneous cell suspension is generated and washed twice withIMDM medium without any supplements, by repeated centrifugation (500 g,5 min) and resuspension steps.

5×10⁶ myeloma cells (Sp2/0 Köhler and Milstein, Eur. J. Immunol., 6, p.511-519, 1976; or X63Ag8.653 Kearney et al., J. Immunol., 123, p.1548-1550, 1979) and 5×10⁷ spleen cells (or 5×10⁷ in vitrodifferentiated plasma cells) are combined, mixed and centrifuged againat 500 g for 5 min. 1 ml of a 50% polyethylene glycol (PEG) 1500dilution in IMDM medium is slowly added to the cell pellet, while thecells are resuspended by tapping. After 2 minutes incubation, 10 ml ofDMEM medium without supplements are slowly added, and cells areeventually centrifuged at 500 g for 5 min. After removal of thesupernatant, the cell pellet is resupended in 1 liter of IMDM basedstroma cell growth medium (described in example 1), containing 100 U/mlrecombinant IL6 and 1× concentrated HAT supplement. The cells aredistributed in 50 96-well tissue culture plates and are incubated at 37°C. until individual hybridoma clones can be identified and isolated forfurther expansion and characterization in tissue culture.

1. A method for the generation of vertebrate lymphocytes that can beused for the production of any heterologous antibody, antigen receptor,artificial binding protein, or functional fragment(s) thereof,comprising the steps of: (a) genetically modifying vertebrate precursorlymphocytes, which (i) are derived from primary lymphoid organs, and(ii) have the potential to differentiate into mature lymphoid lineagecells, by introducing at least one exogenous genetic element encoding atleast one heterologous antibody, antigen receptor, artificial bindingprotein, or functional fragment thereof; and (b) effectingdifferentiation of said genetically modified precursor lymphocytes intomature lymphoid lineage cells either in vitro or in vivo, therebygenerating lymphocytes capable of producing said heterologous antibody,antigen receptor, artificial binding protein or functional fragment(s)thereof.
 2. The method according to claim 1, further comprising the stepof immortalizing the differentiated lymphocytes by: (a) fusing the sameto myeloma cells for the generation of hybridoma cells; (b) infectingthe same with transforming viruses; or (c) transfecting the same with avector construct ensuring expression of at least one transformingoncogene; thereby generating vertebrate lymphocytes capable ofpermanently producing said heterologous antibody, antigen receptor,artificial binding protein, or functional fragment(s) thereof.
 3. Themethod according to claim 1, wherein the vertebrate precursorlymphocytes are able to express at least one component of the lymphoidV(D)J recombination machinery and originate from jawed vertebratescomprising cartilaginous fish, bony fish, amphibians, reptilia, birds,mammals including pigs, sheep, cattle, horses and rodents includingmice, rats, rabbits and guinea pigs, with murine precursor (pre) Blymphocytes from mice being preferred.
 4. The method according to claim1, wherein said vertebrate precursor lymphocytes are deprived of theirpotential to express endogenous antibodies and/or antigen receptors orfunctional fragment(s) thereof, which is achieved by isolating/selectingvertebrate precursor lymphocytes being deficient in expressingendogenous immunoglobulins or fragments thereof, and/or by introducinginto said vertebrate precursor lymphocytes at least one vector constructdesigned to functionally inactivate at least one allele of at least onegenetic element, which is selected from the group consisting of: (a) thecoding regions of the immunoglobulin heavy chain gene locus, includingall or parts of the V, D, and J gene segments, and any of the codingregions for the constant region exons for μ, δ, γ, ε, and α heavychains, with or without their membrane spanning exons; (b) the codingregions of the immunoglobulin κ and/or λ light chain gene loci,including any of the V and J gene segment coding regions, as well as anyof the constant region exons; (c) the coding regions of the T cellreceptor α, β, γ, and δ gene loci, including all or parts of the V, Dand J gene segments, and any of the coding regions for the α, β, γ, andδ constant region exons; (d) the cis-acting immunoglobulin heavy chaingene locus enhancer elements, including the heavy chain intron enhancerand the 3′α enhancer; (e) the cis-acting immunoglobulin light chain genelocus enhancer elements, including the κ light chain intron enhancer(κiE), the 3′κ enhancer, and the λ2-4 and λ3-1 enhancers; (f) thecis-acting T cell receptor gene loci enhancer elements, including theTCR α, β, γ, and δ enhancers; (g) the trans-acting recombinationactivating genes, RAG-1 and RAG-2, including their promoter and enhancerelements, as well as their coding regions; and (h) the trans-acting DNArepair genes essential for V(D)J recombination, including Ku70, Ku86,the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), DNAligase IV, XRCC4 and Artemis, including their promoter and enhancerelements, as well as the coding regions of said genes.
 5. The methodaccording to claim 4, wherein said vector constructs include genetargeting vectors comprising regions of DNA sequence homology to said atleast one genetic element enabling for homologous recombination,preferably flanking a positive selection marker enabling selection ofpositive transfectants.
 6. The method according to claim 5, wherein saidgene targeting vectors additionally comprise a pair of DNA recognitionsequences for site-specific DNA recombination enzymes, flanking thepositive selection marker, enabling deletion of said positive selectionmarker upon transfection and transient expression of nucleic acidsequences encoding at least one of the cognate recombinase enzymes. 7.The method according to claim 5, wherein said gene targeting plasmidvectors additionally comprise a negative selection marker enablingselection against transfectants in which said gene targeting vectors arerandomly integrated into the genome by non-homologous recombination. 8.The method according to claim 1, wherein said at least one exogenousgenetic element encoding a heterologous antibody, an artificial bindingprotein, an antigen receptor, or (a) functional fragment(s) thereof, iscarried on a genetic construct selected from the group consisting of:(a) recombinant retroviral DNA constructs comprising promoter, enhancerand coding nucleic acid sequences operably linked to allow expression ofat least one heterologous antibody, artificial binding protein, antigenreceptor, or functional fragment thereof, being either wild-type orhaving (a) designed mutation(s) in the primary amino acid sequence(s) orbeing artificial; (b) recombinant plasmid-based DNA constructscomprising promoter, enhancer and coding nucleic acid sequences operablylinked to allow expression of at least one heterologous antibody,artificial binding protein, antigen receptor, or functional fragmentthereof, being either wild-type or having (a) designed mutation(s) inthe primary amino acid sequence(s) or being artificial; (c) recombinantplasmid-based mini-immunoglobulin or T cell receptor gene loci withunrearranged V, D and J gene segments operably linked to allow V(D)Jrecombination and subsequent expression of at least one heterologousantibody or T cell receptor, or functional fragment thereof, beingeither wild-type or having (a) designed mutation(s) in the primary aminoacid sequence(s); (d) bacterial, yeast or vertebrate artificialchromosomes comprising parts or all of immunoglobulin or T cell receptorgene loci in germline configuration operably linked to allow V(D)Jrecombination and subsequent expression of at least one heterologousantibody or T cell receptor, or functional fragment thereof, beingeither wild-type or having (a) designed mutation(s) in the primary aminoacid sequence(s); (e) bacterial, yeast or vertebrate artificialchromosomes comprising parts or all of at least one heterologousimmunoglobulin or T cell receptor gene locus in modified arrangementdesigned to allow (D)J recombination and subsequent expression of atleast one heterologous antibody or T cell receptor, or functionalfragment thereof, being either wild-type or having (a) designedmutation(s) in the primary amino acid sequence(s); (f) trans-chromosomeelements which are fragments of heterologous chromosomes harbouringparts or all of immunoglobulin or T cell receptor gene loci in germlineconfiguration allowing V(D)J recombination and subsequent expression ofat least one heterologous antibody or T cell receptor, being wild-typewith respect to the primary amino acid sequence(s).
 9. The methodaccording to claim 1, wherein the at least one exogenous genetic elementencodes a native or modified human antibody, a human binding protein, ahuman antigen receptor, or (a) functional fragment(s) thereof.
 10. Themethod according to claim 1, wherein the at least one exogenous geneticelement encodes a heterologous or artificial receptor capable ofundergoing affinity maturation of the binding region.
 11. The methodaccording to claim 1, wherein the differentiation of vertebrateprecursor lymphocytes is effected in vitro by: (a) arrestingproliferation of said vertebrate precursor lymphocytes and inducingdifferentiation into mature lymphocyte lineage cells by cultivating inthe absence of any precursor lymphocyte growth factor; and (b) inducingterminal lymphocyte differentiation by further cultivating said cells inthe presence of at least one of the following components selected from:(i) soluble T cell related stimulating factors, comprisinginterleukin-2, interleukin-4, interleukin-5, interleukin-6,interleukin-10, interleukin-13, TGF-β, and IFN-γ; (ii) factorsactivating co-stimulatory receptors of B cells, comprising agonisticantibodies or active, recombinant ligands specific for CD40, B7-1(CD80), B7-2 (CD86), complement receptors 1 (CD35) and 2 (CD21), LFA-1(CD11 a), LFA-3 (CD58), CD19, CD20, CD30, CD32, CD37, CD38, CD70, CD71,Iga (CD79α), Igβ(3 (CD79β), TAPA-1 (CD81), Fas (CD95), TNF-receptor1(p55, CD120α), TNF-receptor2 (p75, CD120β), Ox-40 (CD134), andlymphotoxin-/3 receptor; and (iii) B cell mitogenic factors, T cellindependent antigens of type 1, and other polyclonal activators,including lipoplysaccharide (LPS), lipoproteins from gram negativebacteria, polyanions, poly-dIdC, pokeweed mitogen (PWM), andanti-immunoglobulin reagents; and combinations thereof.
 12. The methodaccording to claim 1, wherein said differentiation in vivo is effectedupon transplantation of said genetically modified precursor lymphocytesinto a suitable vertebrate host.
 13. The method according to claim 12,wherein said lymphocytes are co-transplanted into said host with naiveor antigen primed T helper lymphocytes.
 14. The method according toclaim 12, wherein said differentiation in vivo is followed byimmunization of said host with at least one desired immunogenic compoundor composition.
 15. The method according to claim 12, wherein saidvertebrate host is a compatible host being deficient with respect to thegeneration of endogenous B cells, T cells, and/or NK (natural killer)cells, or a combination thereof.
 16. The method according to claim 12,wherein the vertebrate host is selected from jawed vertebratescomprising cartilaginous fish, bony fish, amphibians, reptilia, birds,and mammals including pigs, sheep, cattle, horses and rodents includingmice, rats, rabbits and guinea pigs, with mice being the preferred hostspecies.
 17. A method comprising the steps of: (i) obtaining geneticallymodified and differentiated vertebrate lymphocytes by the methodaccording to claim 1, and (ii) using the lymphocytes to produce aheterologous antibody, artificial binding protein, antigen receptor, orfunctional fragment(s) thereof.
 18. The method according to claim 17,wherein the method according to claim 1 is followed by the steps of: (a)isolating from said differentiated lymphocytes the at least oneexogenous genetic element, and (b) placing said genetic element(s) in acontext enabling production of said at least one heterologous antibody,antigen receptor, artificial binding protein, or functional fragment(s)thereof.
 19. The method according to claim 17, wherein said heterologousantibody, artificial binding protein, antigen receptor, or functionalfragment(s) thereof, displays one unique specificity and is thereforemonoclonal.
 20. The method according to claim 17, wherein saidheterologous antibody, artificial binding protein, antigen receptor, orfunctional fragment(s) thereof, displays more than one uniquespecificity and is therefore polyclonal.
 21. The method according toclaim 17, wherein said heterologous antibody, artificial bindingprotein, antigen receptor, or functional fragment(s) thereof, completelyor partially resembles a human antibody, antigen receptor, or bindingprotein, as can be assembled on the basis of the human geneticrepertoire or parts thereof.
 22. The method according to claim 17,wherein the heterologous antibody, artificial binding protein, antigenreceptor, or functional fragment(s) thereof to be produced is selectedfrom the group consisting of: (a) antibodies being either membrane boundor secreted, and consisting of both heterologous heavy and light chainpolypeptides in the stoichiometric composition found in naturalantibodies and consisting of any of the known heavy (μ, δ, γ, α, ε)and/or light (κ and λ) chain isotypes; (b) antibodies with combinationsof heavy and light chain polypeptides being completely human withrespect to the primary amino acid sequence; (c) hybrid antibodiescontaining heterologous heavy or light chain polypeptides from differentvertebrate species; (d) secreted Fab, scFv and F(ab′)₂ antibodyfragments being either completely or partially heterologous; (e)fragments of antibodies covalently coupled via linker peptides,resulting in bispecific or multispecific antibody fragments; (f) T cellreceptors of the α, β, γ, and δ isotype, antigen receptors, and otherbinding proteins with structural resemblance to proteins of theimmunoglobulin superfamily; and functional fragments thereof. 23.Genetically modified vertebrate precursor lymphocytes and more maturelymphoid lineage cells derived therefrom, obtainable by a methodaccording to claim
 1. 24. Immortalized cells producing heterologousantibodies, artificial binding proteins, antigen receptors, orfunctional fragments thereof, obtainable according to claim
 2. 25.Vector constructs for use in a method according to claim
 4. 26. Geneticconstruct for use in the method according to claim
 8. 27. Pharmaceuticalor diagnostic preparation, comprising at least one antibody, artificialbinding protein, antigen receptor, or functional fragment thereof,obtained by a method according to claim 17, displaying either wild-typeimmune effector functions, or modified or artificial effector functionsnot derivable from germline encoded heterologous immunoglobulins orantigen receptors.